Evaluation of a Clostridium difficile infection management policy with clinical pharmacy and medical microbiology involvement at a major Canadian teaching hospital.

WHAT IS KNOWN AND OBJECTIVE: Clostridium difficile infection (CDI) represents a spectrum of disease and is a significant concern for healthcare institutions. Our study objective was to assess whether implementation of a regional CDI management policy with Clinical Pharmacy and Medical Microbiology and Infection Control involvement would lead to an improvement in concordance in prescribing practices to an evidence-based CDI disease severity assessment and pharmacological treatment algorithm. METHODS: Conducted at a tertiary care teaching hospital, this two-phase quality assurance study consisted of a baseline retrospective healthcare record review of patients with CDI prior to the implementation of a regional CDI management policy followed by a prospective evaluation post-implementation. RESULTS AND DISCUSSION: One hundred and forty-one CDI episodes in the pre-implementation group were compared to 283 episodes post-implementation. Overall treatment concordance to the CDI treatment algorithm was achieved in 48 of 141 cases (34%) pre-implementation compared with 136 of 283 cases (48·1%) post-implementation (P = 0·01). The median time to treatment with vancomycin was reduced from five days to one day (P < 0·01), with median length of hospital stay decreasing from 30 days to 21 days (P = 0·01) post-implementation. There was no difference in 30-day all-cause mortality. WHAT IS NEW AND CONCLUSION: A comprehensive approach with appropriate stakeholder involvement in the development of clinical pathways, education to healthcare workers and prospective audit with intervention and feedback can ensure patients diagnosed with CDI are optimally managed and prescribed the most appropriate therapy based on CDI disease severity.

Meta-analysis in microbiology.

The use of meta-analysis in microbiology may facilitate decision-making that impacts public health policy. Directed at clinicians and researchers in microbiology, this review outlines the steps in performing this statistical technique, addresses its biases and describes its value in this discipline. The survey to estimate extent of the use of meta-analyses in microbiology shows the remarkable growth in the use of this research methodology, from a minimal Asian output to a level comparable with those of Europe and North America in the last 7 years.

Enteroaggregative Escherichia coli and acute diarrhea in children: a meta-analysis of South Asian populations.

The purpose of this investigation was to evaluate the association of enteroaggregative Escherichia coli (EAEC) with acute diarrhea in children of South Asian populations. Our meta-analysis included 18 studies published between 1989 and 2011. The odds ratio (OR) was used to evaluate all available observational epidemiology studies. Modifying effects on the overall OR were approached with outlier, subgroup, cumulative, and cumulative recursive analyses. Synthesis of the 18 observational studies revealed an association between EAEC carriage and acute diarrhea, with an overall OR of 1.51, which was significant (p = 0.008), heterogeneous (Pheterogeneity < 0.0001), and unaffected by outlier analysis. This analysis, however, affected the subgroups by eliminating the following: (i) heterogeneity (from Pheterogeneity < 0.0001 to 0.30-0.72) of pooled ORs in the underpowered (OR 1.37, p = 0.15), Indian (OR 1.92, p = 0.09), and hospital-based (OR 1.66, p = 0.06) studies; (ii) non-significance of these three subgroups (OR 1.56-2.01, p < 0.0001-0.003); (iii) significance of the high-powered studies (from OR 1.70, p = 0.02 to OR 1.15, p = 0.28); (iv) heterogeneity (from Pheterogeneity < 0.0001-0.0002 to 0.11-0.15) of pooled ORs in period three (OR 1.85, p = 0.14), population-based (OR 1.36, p = 0.09), and pCVD432 (OR 1.53, p = 0.07) studies. In general, outlier treatment increased precision with the narrowing of confidence intervals, overall, and in the subgroups. Cumulative meta-analysis generally resulted in increases in the frequencies of significant effects and of heterogeneity. This meta-analysis on observational studies suggests that the association between EAEC and acute diarrhea in children is that of increased risk. This effect generally comes from heterogeneous studies of South Asian populations, but is modified with outlier and subgroup treatments.

Role of inducible cyclooxygenase and prostaglandins in Clostridium difficile toxin A-induced secretion and inflammation in an animal model.

Cyclooxygenase (Cox)-2 expression and inhibition were investigated in a rabbit ileal loop model of Clostridium difficile colitis and diarrhea. Intestinal tissue stimulated with C. difficile toxin A showed up-regulation of Cox-2 expression in lamina propria macrophages and elevated prostaglandin levels. Toxin A-stimulated loops exhibited severe inflammation and increased secretory volume. Celecoxib, a specific Cox-2 inhibitor, significantly reduced toxin A-induced prostaglandin production. Furthermore, celecoxib (> or =0.02 mg/mL) blocked both histologic damage (mean histologic grade, 1.25 vs. 3.44 in rabbits receiving toxin A alone; P<.0005) and secretion (volume:length ratio, 0.18 vs. 0.72 in those receiving toxin A alone; P=.002) in toxin A-stimulated loops in a dose-related manner. Thus, toxin A induced expression of Cox-2 in the host, and prostaglandins produced through Cox-2 were involved in the mediation of the increased secretion of electrolytes and water and the inflammatory response induced by toxin A.

Enteroaggregative Escherichia coli expresses a novel flagellin that causes IL-8 release from intestinal epithelial cells.

Enteroaggregative Escherichia coli (EAEC) is an emerging cause of acute and persistent diarrhea worldwide. EAEC infections are associated with intestinal inflammation and growth impairment in infected children, even in the absence of diarrhea. We previously reported that prototype EAEC strains rapidly induce IL-8 production by Caco-2 intestinal epithelial cells, and that this effect is mediated by a soluble, heat-stable factor released by these bacteria in culture. We herein report the cloning, sequencing, and expression of this biologically active IL-8-releasing factor from EAEC, and its identification as a flagellin that is unique among known expressed proteins. Flagella purified from EAEC 042 and several other EAEC isolates potently release IL-8 from Caco-2 cells; an engineered aflagellar mutant of 042 does not release IL-8. Finally, cloned EAEC flagellin expressed in nonpathogenic E. coli as a polyhistidine-tagged fusion protein maintains its proinflammatory activity. These findings demonstrate a major new means by which EAEC may cause intestinal inflammation, persistent diarrhea, and growth impairment that characterize human infection with these organisms. Furthermore, they open new approaches for diagnosis and vaccine development. This novel pathogenic mechanism of EAEC extends an emerging paradigm of bacterial flagella as inflammatory stimuli.

Failure of amphotericin B colloidal dispersion in the treatment of paracoccidioidomycosis.

Although amphotericin B desoxycholate is considered the most effective treatment for disseminated Paracoccidioides brasiliensis infections, little is known about the efficacy of lipid-based formulations of amphotericin B in this infection. In this study, we treated four adults with the juvenile form of paracoccidioidomycosis with 3 mg/kg/day of amphotericin B colloidal dispersion for at least 28 days. Although all of the patients initially responded by clinical observation, all four patients relapsed within six months. The use of amphotericin B colloidal dispersion for the initial induction of paracoccidioidomycosis failed to cure this infection. Possible reasons for failure include dose, duration, or reduced efficacy of this lipid preparation. For many fungal infections, lipid-based preparations have been shown to have a therapeutic-toxic advantage, but our experience with Paracoccidioides infections suggests that more careful studies will need to be performed before they can be recommended for use in this mycosis.

How intestinal bacteria cause disease.

An improved understanding of how intestinal bacteria cause disease has become increasingly important because of the emergence of new enteric pathogens, increasing threats of drug resistance, and a growing awareness of their importance in malnutrition and diarrhea. Reviewed here are the varied ways that intestinal bacteria cause disease, which provide fundamental lessons about microbial pathogenesis as well as cell signaling. Following colonization, enteric pathogens may adhere to or invade the epithelium or may produce secretory exotoxins or cytotoxins. In addition, by direct or indirect effects, they may trigger secondary mediator release of cytokines that attract inflammatory cells, which release further products, such as prostaglandins or platelet-activating factor, which can also trigger secretion. An improved understanding of pathogenesis not only opens new approaches to treatment and control but may also suggest improved simple means of diagnosis and even vaccine development.

Intestinal secretory factor released by macrophages stimulated with Clostridium difficile toxin A: role of interleukin 1beta.

Clostridium difficile toxin A is associated with enterocolitis in animals and humans. However, the mechanisms of its secretory and damaging effects are not totally understood. In this work, we examined the intestinal secretion of electrolytes and water caused by supernatants from macrophages stimulated with toxin A in rabbit ileal mucosa mounted in Ussing chambers. We also investigated the mechanism by which the intestinal secretory factor (ISF) is released from stimulated macrophages. Supernatants from macrophages stimulated with toxin A caused potent intestinal secretion (change in short-circuit current [DeltaIsc], 76 microA x cm-2; P < 0.01). The release of the ISF was pertussis toxin sensitive (reduction, 61%; P < 0.01) and was also reduced (P < 0.05) by a protein synthesis inhibitor (67%), protease inhibitors (57%), a phospholipase A2 inhibitor (54%), a cyclo-oxygenase inhibitor (62%), a dual cyclo- and lipoxygenase inhibitor (48%), a platelet-activating factor (PAF) receptor antagonist (55%), and tumor necrosis factor alpha (TNF-alpha) synthesis inhibitors (48%). However, this release was not inhibited by a lipo-oxygenase inhibitor. Monoclonal anti-interleukin 1beta (IL-1beta) but not anti-IL-1alpha antibody blocked (72%; P < 0.01) the secretory action of the ISF, as did recombinant human IL-1 receptor antagonist (80%; P < 0.01). High levels of IL-1beta (3,476 pg/ml) were detected by an enzyme-linked immunosorbent assay in the above supernatants. Furthermore, the addition of IL-1beta to the serosal side caused a potent secretory effect (DeltaIsc, 80 microA x cm-2; P < 0.01). These results show that macrophages stimulated with toxin A release an ISF capable of provoking intestinal secretion. The regulation of this factor is dependent upon the activation of the G protein. In addition, prostaglandins, PAF, and TNF-alpha are involved in the release of the ISF. We conclude that IL-1beta is probably the ISF released by macrophages in response to toxin A.

Enteroaggregative Escherichia coli produce intestinal inflammation and growth impairment and cause interleukin-8 release from intestinal epithelial cells.

Enteroaggregative E. coli (EAggEC) are emerging as an important cause of persistent diarrhea, especially in children in the developing world, yet the pathogenesis of EAggEC infection is poorly understood. In an ongoing prospective study of childhood diarrhea in an urban Brazilian slum, EAggEC are the leading cause of persistent diarrhea. Children from this study with EAggEC and persistent diarrhea had significant elevations in fecal lactoferrin, interleukin (IL)-8, and IL-1beta. Moreover, children with EAggEC without diarrhea had elevated fecal lactoferrin and IL-1beta concentrations. The children with EAggEC in their stool had significant growth impairment after their positive culture, regardless of the presence or absence of diarrhea. Finally, 2 EAggEC strains were shown to cause IL-8 release from Caco-2 cells, apparently via a novel heat-stable, high-molecular-weight protein. These findings suggest that EAggEC may contribute to childhood malnutrition, trigger intestinal inflammation in vivo, and induce IL-8 secretion in vitro.

Protozoal agents: what are the dangers for the public water supply?

Safe and efficient treatment of drinking water has been one of the major public health advances of the twentieth century. People in developed countries generally take for granted that their water is safe to drink, a luxury the majority of the world's population does not have. The leading cause of infant mortality in the developing world is infectious diarrhea, and the prevalence of diarrheal pathogens is largely influenced by the quality and quantity of clean water available for drinking and washing. Until recently, modern water treatment had all but eliminated these concerns in developed nations. Over the past two decades, however, the safety of our water supply has been threatened by the emergence of Cryptosporidium parvum, a protozoal pathogen. The hearty oocysts of this organism survive chlorination and filtration to cause a diarrheal illness that, while unpleasant enough in healthy people, is devastating in immunocompromised individuals. The 1993 Milwaukee outbreak, in which 403,000 people developed diarrhea from drinking water that met all the updated federal safety standards, demonstrated the tremendous public health importance of this organism. While earlier attention had focused on Giardia and amebic infections, the other "emerging" protozoan besides Cryptosporidium is Cyclospora. This review discusses the protozoal pathogens, including Cryptosporidium parvum, Giardia lamblia, Entamoeba histolytica, and Cyclospora cayetanensis, that cause waterborne diarrheal outbreaks and the threats they pose to the public.

Fecal lactoferrin, interleukin-1beta, and interleukin-8 are elevated in patients with severe Clostridium difficile colitis.

Twenty-two patients with Clostridium difficile colitis as determined by positive enzyme immunoassay for toxin A were evaluated for fecal inflammatory markers and their relationship to the severity of illness. Fourteen of 22 specimens were positive for fecal lactoferrin (FLF), with titers from 1:50 to 1:800. Nine of 10 stools tested had ratios of interleukin-1beta (IL-1beta) to IL-1 receptor antagonist (IL-1ra) of >0.01. Seventeen of 22 specimens also had elevated IL-8 concentrations, and 12 of 14 had elevated IL-1beta concentrations. A review of the 18 available patient records revealed that fecal IL-8 concentrations, IL-1beta/IL-1ra ratios, and FLF titers were significantly higher in patients with moderate to severe disease than in patients with mild disease. These findings suggest that the proinflammatory effects of C. difficile may directly influence clinical characteristics of human disease.

New evidence for an inflammatory component in diarrhea caused by selected new, live attenuated cholera vaccines and by El Tor and Q139 Vibrio cholerae.

Using a lactoferrin latex agglutination assay, we have compared the inflammatory responses to a cholera vaccine candidate, CVD 110, in which all known toxin genes have been deleted or mutated yet still produced significant diarrhea, with a less reactive vaccine strain and wild-type El Tor and 0139 Vibrio cholerae strains. Data suggest that diarrhea due to attenuated and wild-type El Tor V. cholerae, and to a lesser extent 0139 V. cholerae, involves an inflammatory response. Further study is required to further elucidate the mechanism of the process(es) involved.

Secretin receptors mediating rat forestomach relaxation.

Frozen sections of the rat stomach were incubated with 125I-labeled porcine secretin, and then secretin binding sites were localized by autoradiography. Saturable binding was observed only in the muscularis externa (circular and longitudinal smooth muscle layers) of the proximal nonglandular forestomach. Saturable binding was quantitated by densitometry. 125I-porcine secretin bound to a single class of high-affinity binding sites with a dissociation constant of 0.6 nM. Porcine and rat secretins were nearly equipotent in inhibiting saturable 125I-porcine secretin binding, and vasoactive intestinal polypeptide, peptide histidine-isoleucine, and glucagon were much weaker. Carbachol (100 microM) stimulated a sustained increase in tension in forestomach muscle in vitro, and porcine secretin caused relaxation of this stimulated contraction. We conclude that rat forestomach smooth muscle expresses a high-affinity specific secretin binding site that mediates relaxation. This putative secretin receptor may mediate some of the actions of secretin on gastric motility.