Cytokine production in patients with anorexia nervosa, bulimia nervosa, and obesity.

OBJECTIVE: We previously reported elevated serum levels of the cytokines interleukin-6 (IL-6) and transforming growth factor-beta (TGF-beta) in patients with anorexia nervosa (AN). We investigated the cellular production of these two cytokines and of interferon-gamma (IFN-gamma), interleukin-1alpha (IL-1alpha), and tumor necrosis factor-alpha (TNF-alpha) in subjects with AN, bulimia nervosa (BN), and obesity as well as in normal-weight control subjects. METHODS: Supernatant fluids from isolated peripheral blood mononuclear cells (PBMC) incubated with and without concanavalin A (ConA) were assayed for cytokine concentrations by enzyme-linked immunosorbent assay (ELISA). RESULTS: Significant differences across the four groups were found in the stimulated cellular production of IFN-gamma and IL-6. Stimulated IFN-gamma production was elevated in the AN group compared to controls. IL-6 production was significantly elevated in obese subjects relative to the two normal-weight groups, BN and controls, and tended to be higher in the AN group than in the controls, but not significantly so. IL-1alpha production was greater in obese subjects. CONCLUSION: The findings of increased IFN-gamma production and a tendency toward increased IL-6 production (both of which suppress food intake in animals) in individuals who severely restrict food intake suggest a potential role for these cytokines in the pathogenesis of AN. Elevated IL-6 and IL-1alpha production by PBMC in obese individuals requires further investigation to determine if these cytokines contribute to the development or perpetuation of obesity.

Measuring delirium severity in older general hospital inpatients without dementia. The Delirium Severity Scale.

The authors evaluated the validity, reliability, and sensitivity to change of the Delirium Severity Scale (DSS), a 10-minute assessment consisting of Forward Digit Span and Similarities. Twenty-two older inpatients with delirium but not dementia and 15 control patients were administered the DSS during hospitalization. Scores were significantly inversely correlated with experts' quantitative ratings of severity at all three time-points examined. The DSS showed significant improvement over time (P < 0.001) and significant correlation with improvement in expert ratings (P = 0.026). The DSS shows promise as a valid and reliable measure sensitive to changing symptom severity.

The association of serum anticholinergic activity with delirium in elderly medical patients.

To investigate the hypothesis that elevated serum anticholinergic activity is independently associated with delirium in ill elderly persons, the authors performed a cross-sectional study of 67 acutely ill older medical inpatients. The presence of delirium was evaluated with the Confusion Assessment Method, and the presence of many delirium symptoms was measured by the Delirium Symptom Interview. Demographic data and clinical characteristics that may be important for the development of delirium were also collected. Logistic regression techniques demonstrated that elevated serum anticholinergic activity was independently associated with delirium. Among the subjects with delirium, a greater number of delirium symptoms was associated with higher serum anticholinergic activity.

Serum anticholinergic activity and behavior following atropine sulfate administration in the rat.

Anticholinergic agents such as atropine and scopolamine have long been suggested to produce delirium-like states in humans and experimental animals. Evidence for an anticholinergic mechanism in the pathogenesis of human delirium has accumulated, leading to studies of the behavioral effects of the anticholinergic drug atropine in animals. The current study addresses the adequacy of animal models of delirium in terms of sensitivity, specificity and pharmacological relevance. A multiple fixed-ratio fixed-interval reinforcement schedule was used to test the effects of relatively low doses of atropine on behavior in rats. Additionally, total serum anticholinergic activity (SAA) was measured under dose and time course conditions identical to those used in the behavioral study. Atropine reduced high and low rates of responding in a dose-dependent manner, and SAA increased in a dose dependent manner. SAA at atropine doses of 0.1 mg/kg to 1.0 mg/kg was similar to that found in delirious humans. These behavioral and serum level data suggest that relatively low doses of atropine, substantially below those used in previous attempts to model delirium using rats, may be more pharmacologically relevant to delirium and may minimize non-specific peripheral effects of this drug.

Protein-losing enteropathy is associated with Clostridium difficile diarrhea but not with asymptomatic colonization: a prospective, case-control study.

A prospective, case-control study was performed in which enteric protein loss and nutritional status were measured in patients with symptomatic and asymptomatic infections due to Clostridium difficile. Enteric protein loss, measured by elevated levels of fecal alpha1-antitrypsin, was detected in 14 of 20 cases and controls with diarrhea (9 of 10 cases with C. difficile-associated diarrhea and 5 of 10 age-matched control with diarrhea not associated with C. difficile) compared with none of 20 asymptomatic cases and controls (10 colonized cases and 10 noncolonized controls without diarrhea who were matched by age and clinical diagnosis) (P < .0001). Cases and controls with diarrhea had higher prognostic nutritional index values (P = 0.005) and lower levels of serum albumin, transferrin, and cholesterol than did the asymptomatic cases and controls. Decreased nutritional status, measured by increased prognostic nutritional index values, was associated with the presence of diarrhea but not with the presence of C. difficile. Protein-losing enteropathy was associated with C. difficile only in the presence of diarrhea, and we did not detect an increased risk of protein-losing enteropathy or malnutrition as a consequence of asymptomatic colonization with C difficile.

Effectiveness of liquid soap vs. chlorhexidine gluconate for the removal of Clostridium difficile from bare hands and gloved hands.

OBJECTIVE: To compare liquid soap versus 4% chlorhexidine gluconate in 4% alcohol for the decontamination of bare or gloved hands inoculated with an epidemic strain of Clostridium difficile. DESIGN: C difficile (6.7 log10 colony-forming units [CFU], 47% spores), was seeded onto bare or latex gloved hands of ten volunteers and allowed to dry. Half the volunteers initially washed with soap and half with chlorhexidine, followed by the other agent 1 week later. Cultures were done with Rodac plates at three sites on the hand: finger/thumbtips, the palmar surfaces of the fingers, and the palm. Statistical comparison was by paired Student's t test. RESULTS: On bare hands, soap and chlorhexidine did not differ in residual bacterial counts on the finger/thumbtips (log10 CFU, 2.0 and 2.1, P = NS) and fingers (log10 CFU, 2.4 and 2.5, P = NS). Counts were too high on bare palms to quantitate. On gloved hands, soap was more effective than chlorhexidine on fingers (log10 CFU 1.3 and 1.7, P < .01) and palms (log10 CFU 1.5 and 2.0, P < .01), but not finger/thumbtips (log10 CFU 1.6 with each, P = NS). Residual C difficile counts were lower on gloved hands than bare hands (P < 0.01 to < 0.0001). CONCLUSIONS: The two agents did not differ significantly in residual counts of C difficile on bare hands, but on gloved hands residual counts were lower following soap wash than following chlorhexidine wash. These observations support the use of either soap or chlorhexidine as a handwash for removal of C difficile, but efficacy in the prevention of C difficile transmission must be determined by prospective clinical trials.

Wide diversity of Clostridium difficile types at a tertiary referral hospital.

Nosocomial Clostridium difficile infection was investigated at a hospital with 15 cases of C. difficile diarrhea per 1000 discharges. From January 1991 to May 1991, patients admitted or transferred to five wards or units had weekly rectal swabs taken for culture; in addition, all cytotoxin-positive stools were cultured. Restriction enzyme analysis (REA) was used for molecular typing. Among 205 isolates from 39 patients with C. difficile diarrhea and 67 asymptomatically colonized, 55 distinct REA banding patterns were identified. Evidence for patient-to-patient transmission was limited, in that numerous strains were found even among clustered cases of diarrhea. Patients who acquired C. difficile in the community or other hospitals constituted 32% of culture-positive patients and contributed 44% of the REA types. Diversity of C. difficile strains was in part the result of patients acquiring C. difficile in the community or other hospitals. High incidences of nosocomial C. difficile diarrhea do not necessarily indicate clonal epidemics.

Comparison of restriction endonuclease analysis, ribotyping, and pulsed-field gel electrophoresis for molecular differentiation of Clostridium difficile strains.

A combined clinical and molecular epidemiologic analysis of 46 strains of Clostridium difficile, including 16 nosocomial isolates from one ward (outbreak ward) plus 17 other nosocomial isolates and 13 community-acquired isolates, was performed. HindIII digests of total cellular DNA were analyzed by restriction enzyme analysis (REA) and ribotyping; SmaI digests were analyzed by pulsed-field gel electrophoresis (PFGE). Isolates were assigned to typing groups on the basis of the profiles detected; isolates with closely related profiles were assigned to subgroups. The 16 isolates from the outbreak ward were resolved by both REA and PFGE into five distinct groups; 13 isolates represented two REA groups and three PFGE groups and two isolates were resolved as distinct groups by both techniques. DNA obtained from one isolate was persistently partially degraded, precluding analysis by PFGE. Seventeen sporadic nosocomial isolates were resolved by REA and PFGE into comparable numbers of groups (i.e., nine groups) and subgroups (i.e., 15 and 14 subgroups, respectively), with two isolates not evaluable by PFGE. The 13 epidemiologically unrelated community-acquired isolates were assigned to 11 groups by REA and to 12 groups by PFGE. Overall, ribotyping identified only nine groups among the 46 isolates. We conclude that REA and PFGE have comparable discriminatory powers for epidemiologic typing of C. difficile isolates and that ribotyping is appreciably less discriminatory. For a few isolates, partial DNA degradation prevented analysis by PFGE but not by REA or ribotyping; the cause of the degradation is unknown.

Decrease in nosocomial Clostridium difficile-associated diarrhea by restricting clindamycin use.

OBJECTIVE: To report the investigation and effective control of a nosocomial epidemic of Clostridium difficile-associated diarrhea. DESIGN: Concurrent surveillance for identification of new nosocomial cases, retrospective case-control analysis, and hospital formulary control of antibiotic use. SETTING: University-affiliated Veterans Affairs Medical Center located in southwestern United States. PATIENTS: Hospitalized patients who developed diarrhea submitted stool specimens for cytotoxin assay. Patients who were positive for cytotoxin were compared with control patients without infection. MEASUREMENTS: Isolates of C. difficile were typed by restriction endonuclease analysis. Antimicrobial agent use from hospital pharmacy records and selected patient data from chart review were correlated with frequency of specific laboratory abnormalities. RESULTS: For 13 months, the monthly incidence of C. difficile infection averaged more than five times that for the previous 21 months. Stool specimens from 34 patients (59%) contained a single strain (restriction enzyme analysis type J7). Clindamycin was statistically associated with the epidemic as shown by the following: clindamycin use at our center compared with national normal values, clindamycin use for years before compared with during the epidemic, monthly use of clindamycin compared with monthly frequency of infection, frequency of infection in patients receiving clindamycin compared with that in patients receiving other antimicrobial agents, and amount of clindamycin used by infected patients compared with that used by control patients. Restricting clindamycin use led to a prompt disease in infection rate and the type J7 organisms. CONCLUSION: A nosocomial epidemic of C. difficile diarrhea was controlled by analysis of antibiotic use patterns and by subsequent restriction of clindamycin.

Development of a rapid and efficient restriction endonuclease analysis typing system for Clostridium difficile and correlation with other typing systems.

A HindIII restriction endonuclease analysis (REA) typing system for total genomic Clostridium difficile DNA including a rapid and efficient method of DNA extraction and a scheme for organizing unique electrophoretic DNA band patterns was developed. REA typing was performed by two extraction methods for 1,965 C. difficile isolates obtained from patients with symptomatic C. difficile disease, asymptomatic patients who were C. difficile culture positive, and environmental surfaces. This isolate collection yielded 206 unique REA types, which were organized into 75 groups. A reference strain representing each unique REA type was chosen for DNA band pattern comparisons, cytotoxin testing, and plasmid analysis. The DNA band patterns utilizing a guanidine thiocyanate-EDTA-Sarkosyl DNA extraction method were 94% reproducible, while the original and sporadically problematic diethyl pyrocarbonate-sodium dodecyl sulfate DNA extraction method was 98% reproducible when readable patterns were obtained. Reference strains from 43 of the 75 groups were cytotoxin positive, 28 groups were cytotoxin negative, and 4 groups included both toxigenic and nontoxigenic strains. Cytotoxicity of isolates with a particular REA type was always consistent with the toxicity of the reference strain for that type. REA typing was able to discriminate strain differences within types identified by the immunoblot (89 isolates), bacteriophage-bacteriocin (44 isolates), and ribotyping (23 isolates) methods. REA typing is a sensitive, discriminating, reproducible, and rapid method for differentiating C. difficile strains and is suitable for large-scale epidemiologic studies.

Treatment of asymptomatic Clostridium difficile carriers (fecal excretors) with vancomycin or metronidazole. A randomized, placebo-controlled trial.

OBJECTIVE: To compare the efficacy of vancomycin and metronidazole for eradication of asymptomatic Clostridium difficile fecal excretion as a means of controlling nosocomial outbreaks of C. difficile diarrhea. DESIGN: Randomized, placebo-controlled, non-blinded trial. SETTING: Six hundred-bed regional referral Veterans Affairs Medical Center. PATIENTS: Thirty patients excreting C. difficile without diarrhea or abdominal symptoms. INTERVENTIONS: All patients were randomized to receive 10 days of oral vancomycin, 125 mg four times daily; metronidazole, 500 mg twice daily; or placebo, three times daily. MEASUREMENTS: Stool cultures were obtained during treatment and for 2 months after treatment. All C. difficile isolates were typed by restriction endonuclease analysis (REA). RESULTS: Clostridium difficile organisms were not detected during and immediately after treatment in 9 of 10 patients treated with vancomycin compared with 3 of 10 patients treated with metronidazole (P = 0.02) and 2 of 10 patients in the placebo group (P = 0.005). The fecal vancomycin concentration was 1406 +/- 1164 micrograms/g feces, but metronidazole was not detectable in 9 of 10 patients. Eight of the nine evaluable patients who had negative stool cultures after treatment with vancomycin began to excrete C. difficile again 20 +/- 8 days after completing treatment. Three of these patients received additional antibiotics before C. difficile excretion recurred, and five acquired new C. difficile REA strains. Four of six patients who received only vancomycin before C. difficile excretion recurred were culture-positive at the end of the study compared with one of nine patients who received only placebo (P = 0.047). CONCLUSIONS: Asymptomatic fecal excretion of C. difficile is transient in most patients, and treatment with metronidazole is not effective. Although treatment with vancomycin is temporarily effective, it is associated with a significantly higher rate of C. difficile carriage 2 months after treatment and is not recommended.

Evaluation of cycloserine-cefoxitin-fructose agar and cycloserine-cefoxitin-fructose broth for recovery of Clostridium difficile from environmental sites.

Cycloserine-cefoxitin-fructose agar (CCFA) and cycloserine-cefoxitin-fructose broth (CCFB) containing either 500 or 250 micrograms of cycloserine per ml were compared for efficacy in the isolation of Clostridium difficile from hospital ward environmental sites. A RODAC imprint technique was used to inoculate prereduced CCFA. Moistened swabs were used to inoculate prereduced CCFB from environmental sites immediately adjacent to the RODAC sample sites. CCFA (6% positive) was significantly more sensitive than CCFB (3% positive; P less than 0.005), regardless of the cycloserine concentration. When the CCFA cycloserine concentration was decreased from 500 to 250 micrograms/ml, the overall rate of positive cultures rose from 4 to 17%. Medium containing 500 micrograms of cycloserine per ml may be too inhibitory to isolate many moderately sensitive strains of C. difficile from environmental sites. Regardless of the cycloserine concentration, the CCFA RODAC imprint technique is superior to the CCFB method.

Neutrophil chemotaxis and adherence in vitro and localization in vivo in rabbits with Staphylococcus aureus abscesses.

Decreased neutrophil (PMN) function may contribute to an altered host defense system in hosts with bacterial abscesses but has not been well correlated to in vivo outcome. We have found that blood PMNs from rabbits with chronic (2-week) experimental Staphylococcus aureus abscesses have decreased chemotaxis in response to an S. aureus supernatant (370 +/- 130 microns migration vs 570 +/- 180 microns, p less than 0.05) and decreased adherence (11.5% +/- 13.2% vs 32.9% +/- 18.6%, p less than 0.005) compared with PMNs from animals with acute (24-hour) abscesses. No differences were found in chemokinesis, random migration, and chemotaxis in response to zymosan-activated serum. No plasma inhibitors of PMN chemotaxis or inhibitors of chemotaxins were found. Although animals with chronic abscesses had higher levels of circulating chemotaxins, in both groups of animals abscess chemotaxin levels were greater than the plasma chemotaxin level. Animals with a concomitant chronic abscess had less PMN influx into an acute abscess but also less bacterial growth within the abscess than animals without a concomitant chronic abscess. We conclude that rabbits with chronic staphylococcal abscesses have decreased chemotaxis and adherence measured in vitro and decreased PMN localization in vivo. In this model, these functions were not associated with increased bacterial proliferation in vivo.

Cephalothin clearance of Staphylococcus aureus from two experimental infection sites in the presence and absence of local phagocytic cells.

The clearance of Staphylococcus aureus from perforated peritoneal capsules which are accessible to phagocytic cells, and subcutaneous Visking chambers which exclude phagocytes, was studied simultaneously in eight rabbits implanted with both devices. Animals were treated with cephalothin, 100 mg/kg im every 8 h for sixteen doses, beginning 24 h after inoculation of the infection sites with S. aureus (cephalothin MIC 0.125 mg/l, MBC 0.5 mg/l). At the start of cephalothin, subcutaneous chambers contained a higher concentration of S. aureus (8.4 log10 cfu/ml) than peritoneal capsules (6.8 log10 cfu/ml, P less than 0.001). There was a significant bactericidal effect in subcutaneous chambers on both the third and sixth day of treatment (P less than 0.002), whereas in peritoneal capsules this did not occur until day six. The total reduction in bacterial count in subcutaneous chambers (7.0 log10 cfu/ml) was significantly greater than in capsules (4.4 log10 cfu/ml, P less than 0.002). The mean concentration of cephalothin in subcutaneous chambers (10.7 mg/l) was significantly higher than in peritoneal capsules (6.1 mg/l, P less than 0.02), but no difference in in-vitro killing of S. aureus was detected at these concentrations. We conclude that cephalothin clearance of S. aureus from a site accessible to phagocytes was delayed when compared to a phagocyte-inaccessible site.

Neutrophil localization in acute and chronic experimental abscesses.

Abdominal abscesses are associated with a high mortality, and usually require surgical drainage for cure. A potential mechanism explaining the inability of the host to clear this infection may be in part a result of the inability of the neutrophil to localize at the site of an established infection. To study this question, either acute (4 hours old) or chronic (2 weeks old) abscesses caused by Staphylococcus aureus were created in perforated capsules implanted in the peritoneal cavity of rabbits. Homologous neutrophils were obtained from donor rabbits 4 hours after peritoneal glycogen stimulation and labeled with indium 111 oxine. Only 0.71% of injected 111In-labeled neutrophils localized in the chronic abscesses, compared with 1.77% in acute abscesses (P less than or equal to 0.01). Animals with chronic infections had a lower intravascular recovery of injected neutrophils (P less than 0.002). Failure of neutrophil localization was not associated with less chemotactic activity within the abscess, as measured by a chemotaxis-under-agarose assay, or caused by a barrier surrounding the abscess as detected by radionuclide imaging. Only 0.07% of injected neutrophils localized into acute abdominal abscesses in animals with a concomitant chronic subcutaneous abscess. These chronically infected animals also demonstrated a low peak intravascular recovery of injected neutrophils when compared with animals with only an acute infection (P less than 0.002). These data reveal that neutrophils localize to abscesses poorly in animals with chronic infections. The mechanism is possibly related to a systemic factor(s) associated with a lower intravascular recovery of injected neutrophils in chronically infected animals.

In-111 Pseudomonas aeruginosa: a simple method of labeling live bacteria with a gamma-emitting radioisotope.

We describe a simple and reliable technique for labeling Pseudomonas aeruginosa with a readily available commercial preparation of indium-111 (111In) oxine. Labeling of a heavy bacterial suspension with 500 mu Ci of commercially prepared 111In-oxine resulted in a yield of 0.0004 mu Ci of cell-associated 111In per 10(6) colony-forming units (CFU). The label was 88% bacterially associated and did not effect viability of the organism. Radiolabeling a gram-negative organism with 111In-oxine provides a non-toxic, stable gamma-emitting bacterial tracer.

Relation between in vivo abscess localization and in vitro migration and adherence of neutrophils.

Rabbit peripheral blood and glycogen-stimulated peritoneal neutrophils were labeled with [111In]indium oxine and transfused intravenously into recipient rabbits with experimental abdominal abscesses due to Staphylococcus aureus. Peritoneal neutrophils harvested 4 hr after glycogen infusion localized within the abscesses to a greater extent than did peripheral blood neutrophils (P less than .002). In an in vitro chemotaxis under-agarose assay, peripheral blood neutrophils had greater random migration (P less than .002) and directed migration (P less than .01) than did peritoneal cells. In an in vitro glass slide adherence assay, peritoneal neutrophils were more adherent than were blood neutrophils (P less than .05). The discrepancy between in vivo and in vitro findings may be due to the increased adherence of peritoneal neutrophils. Glycogen-stimulated peritoneal neutrophils have been exposed in vivo to C5a, which is known to decrease migration and increase adherence in vitro of polymorphonuclear neutrophils; consequently, in vivo exposure of neutrophils to C5a may mean in vitro migration data may be misleading in predicting results in vivo.

High-pressure liquid chromatographic assay of ceftizoxime with an anion-exchange extraction technique.

An anion-exchange extraction method was used in conjunction with high-pressure liquid chromatography for assay of ceftizoxime in 181 serum samples. Comparison of this method with bioassay gave a linear regression line described by Y = 1.11 + 0.98 X, with a correlation coefficient of 0.984. The anion-exchange extraction method is a fast, reliable method of preparing serum samples containing ceftizoxime for assay by liquid chromatography.

Inhibition of ribonucleic acid synthesis by group A streptococcal pyrogenic exotoxin.

Group A streptococcal pyrogenic exotoxins (SPEs) A, B, and C and alpha-amanitin enhance host susceptibility to lethal endotoxin shock. The capacity of SPE C and alpha-amanitin to prepare rabbits for the enhancement phenomenon required pretreatment of the animals 1 to 2 h before giving endotoxin. Endotoxin clearance from the circulation of rabbits pretreated with either SPE C or alpha-amanitin was reduced. Even at the time of death, significant amounts of endotoxin remained in the circulation. It is proposed that the SPE and alpha-amanitin inhibit ribonucleic acid synthesis in Kupffer cells with concomitant alteration in reticuloendothelial clearnace function, allowing endotoxin to persist in the circulation and produce host injury. All three SPE types and alpha-amanitin inhibited ribonucleic acid synthesis by 50% or greater in whole liver cells. Kupffer cells, liver cell nuclei, and liver nuclear extracts; inhibition was observed liver cells from both mice and rabbits. The inhibitory effect by SPEs was dose dependent and was observed after as little as 15 min of preincubation with liver cells. The content of ribonucleic acid in liver nuclei of mice pretreated with either SPE C or alpha-amanitan was reduced, whereas total deoxyribonucleic acid and protein content remained unaltered.