Healthcare-associated infections occur commonly in hospitals and have a major impact on patient well-being. The occurrence of the healthcare-associated infection, Clostridium difficile, has been occurring more frequently among hospitalized patients due to an epidemic strain, and is an important cause of antibiotic-associated diarrhea and colitis. This study examined the impact of several organizational factors on the occurrence of C. difficile infection (CDI) in hospitals using an institutional theory perspective. Administrative claims were utilized from University HealthSystem Consortium hospitals to obtain hospital-level data for the calendar year 2011. Data were available for 89 hospitals. Hospital-level analyses, negative binomial regression models, were conducted to test eight developed hypotheses and the associations between organizational factors and the incidence of CDI in hospitals. Cases of CDI were risk-adjusted for known factors associated with CDI. After controlling for factors known to be associated with CDI, the results of the analyses showed that one study hypothesis was supported. That is, hospitals with higher Leapfrog Group Safety Scores had CDI rates that were no different than hospitals with lower Safety Scores. Further, it was found that U.S. News and World Report Best Hospital Honor Roll member hospitals had significantly higher occurrence of CDI as compared to non-Honor Roll member hospitals, though it was predicted that there would be no difference in CDI rates. The organizational factors state-led CDI prevention collaboratives, state mandatory CDI reporting, Magnet status, the rate of central line-associated bloodstream infections and catheter-associated urinary tract infections, and CDI physician champions, were not significantly associated with CDI occurrence.

Abstract not available

Clostridium difficile is a leading cause of nosocomial infection in North America and a considerable challenge to healthcare professionals in hospitals and nursing homes. The Gram-positive bacterium produces two exotoxins, toxin A (TcdA) and toxin B (TcdB), which are the major virulence factors responsible for C. difficile-associated disease (CDAD) and are targets for CDAD therapy. In this work, recombinant single-domain antibody fragments (VHHs) which target the cell receptor binding domains of TcdA or TcdB were isolated from an immune, llama phage display library and characterized. Four VHHs (A4.2, A5.1, A20.1, and A26.8) were potent neutralizers of the cytopathic effects of TcdA in an in vitro assay and the neutralizing potency was enhanced when VHHs were administered in combinations. Epitope mapping experiments revealed that some synergistic combinations consisted of VHHs recognizing overlapping epitopes, an indication that factors other than mere epitope blocking are responsible for the increased neutralization. Binding assays revealed TcdA-specific VHHs neutralized TcdA by binding to sites other than the carbohydrate binding pocket of the toxin. The TcdB-specific VHHs failed to neutralize TcdB, as did a panel of human VL antibodies isolated from a synthetic library. To enhance the stability of the C. difficile TcdA-specific VHHs for oral therapeutic applications, the VHHs were expressed with an additional disulfide bond by introducing Ala/Gly54Cys and Ile78Cys mutations. The mutant VHHs were found to be well expressed, were non-aggregating monomers, retained low nM affinity for TcdA, and were capable of in vitro TcdA neutralization. Digestion of the VHHs with the major gastrointestinal proteases, at biologically relevant concentrations, revealed a significant increase in pepsin resistance for all mutants and an increase in chymotrypsin resistance for the majority of mutants without compromising inherent VHH trypsin resistance. Collectively, the second disulfide not only increased VHH thermal stability at neutral pH, as previously shown, but also represents a generic strategy to increase VHH stability at low pH and impart protease resistance. These are all desirable characteristics for the design of protein-based oral therapeutics. In conclusion, llama VHHs represent a class of novel, non-antibiotic inhibitors of infectious disease virulence factors such as C. difficile toxins.

Clostridium difficile is a leading cause of nosocomial infection in North America and a considerable challenge to healthcare professionals in hospitals and nursing homes. The Gram-positive bacterium produces two exotoxins, toxin A (TcdA) and toxin B (TcdB), which are the major virulence factors responsible for C. difficile-associated disease (CDAD) and are targets for CDAD therapy. In this work, recombinant single-domain antibody fragments (VHHs) which target the cell receptor binding domains of TcdA or TcdB were isolated from an immune, llama phage display library and characterized. Four VHHs (A4.2, A5.1, A20.1, and A26.8) were potent neutralizers of the cytopathic effects of TcdA in an in vitro assay and the neutralizing potency was enhanced when VHHs were administered in combinations. Epitope mapping experiments revealed that some synergistic combinations consisted of VHHs recognizing overlapping epitopes, an indication that factors other than mere epitope blocking are responsible for the increased neutralization. Binding assays revealed TcdA-specific VHHs neutralized TcdA by binding to sites other than the carbohydrate binding pocket of the toxin. The TcdB-specific VHHs failed to neutralize TcdB, as did a panel of human VL antibodies isolated from a synthetic library. To enhance the stability of the C. difficile TcdA-specific VHHs for oral therapeutic applications, the VHHs were expressed with an additional disulfide bond by introducing Ala/Gly54Cys and Ile78Cys mutations. The mutant VHHs were found to be well expressed, were non-aggregating monomers, retained low nM affinity for TcdA, and were capable of in vitro TcdA neutralization. Digestion of the VHHs with the major gastrointestinal proteases, at biologically relevant concentrations, revealed a significant increase in pepsin resistance for all mutants and an increase in chymotrypsin resistance for the majority of mutants without compromising inherent VHH trypsin resistance. Collectively, the second disulfide not only increased VHH thermal stability at neutral pH, as previously shown, but also represents a generic strategy to increase VHH stability at low pH and impart protease resistance. These are all desirable characteristics for the design of protein-based oral therapeutics. In conclusion, llama VHHs represent a class of novel, non-antibiotic inhibitors of infectious disease virulence factors such as C. difficile toxins.

Clostridium difficile has been recognized as one of the leading causes of nosocomial diarrhea and pseudomembranous colitis in human hospitals and nursing homes since the 1970s; however, recent occurrences of community-acquired cases have led researchers to search for additional sources of these infections. Some of the possible sources being investigated include food animals and retail meat. The objective of this study was to compare the prevalence and genotypic characteristics of C. difficile isolated from a closed population in Texas consisting of both humans and swine. Implicit in this objective, we seek to investigate the possible food safety and occupational risks associated with swine and C. difficile. Isolation of C. difficile was performed utilizing an enrichment technique and restrictive media. Polymerase chain reaction (PCR) was used to test for the presence of the toxin A and B genes, the tcdC gene deletion, and the binary toxin gene. Genotypic characteristics were compared using PCR toxinotyping and pulsed-field gel electrophoresis (PFGE). Antimicrobial susceptibility was tested using commercially available tests (ETest®) for 11 different antibiotics. Statistical comparisons (both parametric and non-parametric, and appropriate to the data) were performed both between and among host species. We tested 2,292 aggregated human wastewater samples and 2,936 swine fecal samples from 2004 to 2006 and found 271 (11.8 percent) and 252 (8.6 percent) to be positive for C. difficile, respectively. The prevalence of C. difficile among swine production groups differed significantly (p<0.05); however, prevalence in the human occupational group cohorts (swine workers and non-workers) did not differ (p=0.81). The majority of the human and swine isolates were a PFGE NAP7 (a variant pattern with 90.5 percent similarity) toxinotype V strain. Antimicrobial resistance levels and multi-resistance patterns were generally similar between host species; however, there was decreased susceptibility (p<0.05) to ampicillin, clindamycin, and imipenem observed in swine isolates, whereas there was decreased susceptibility (p<0.05) to ciprofloxacin in the human isolates. The similarity in C. difficile prevalence between swine workers and non-workers suggests a low occupational hazard of working with swine as it relates to C. difficile source. We also found that there is a decreased prevalence of C. difficile in late production groups in swine suggesting a lowered risk of food-borne exposure. However, the majority of the isolates derived from the human wastewater and swine appeared to be of very similar strain types, suggesting that a common environmental point source predominates for both hosts.

Clostridium difficile is the most common infectious cause of nosocomial diarrhea, affecting thousands of patients annually and exacting enormous costs on the U.S. health care system. Early diagnosis is critical to prevent transmission and reduce morbidity and mortality, yet sensitive and specific diagnostic tests with a quick turnaround time are lacking. The objective of this study was to determine if a new commercially available real time polymerase chain reaction (PCR) test would prove more rapid, sensitive and specific than standard methods for the diagnosis of C. difficile infection (CDI). BD GeneOhm Cdiff assay, a real-time PCR assay for detection of C. difficile toxin B (tcdB) gene, was compared with Tox A/B II ELISA and a two-step algorithm which includes C. Diff Chek-60 Glutamate Dehydrogenase (GDH)-antigen assay followed by cytotoxin neutralization. Four-hundred liquid or semisolid stools submitted for diagnostic C. difficile testing were selected: 200 GDH antigen-positive and 200 GDH antigen-negative. All samples were tested by the C. Diff Chek-60 GDH antigen, cytotoxin neutralization, Toxin A/B II ELISA, and BD GeneOhm Cdiff assay. Discrepant specimens were tested by toxigenic culture as an independent gold standard. Chart review was performed on patients with discrepant specimens. As BD GeneOhm Cdiff assay was not FDA-cleared at the time of study, PCR results were not clinically reported. Of 200 GDH-positive samples, 71 were positive by Tox A/B II, 88 were positive by the two-step method, 93 were positive by PCR, and 96 were positive by GDH-antigen only. Of 200 GDH-negative samples, 3 were positive by PCR only. Toxigenic culture was performed on 41 samples with discrepant results and 39 were culture-positive. After culture resolution of discrepants, Tox A/B II detected 70 (66.7%), the two-step method detected 87 (82.9%), and PCR detected 96 (91.4%) of 105 true positives. The BD Gene-Ohm Cdiff assay was more sensitive in detecting toxigenic C. difficile than Tox A/B II (p <0.0001); however, the difference between PCR and the two-step method was not significant (p=0.1237). The BD GeneOhm Cdiff assay took a similar amount of time to perform as the Tox A/B II and was more rapid than the two-step method. Chart review revealed that 18 patients with cytotoxin-negative, PCR-positive discrepant samples were given 1-2 days of therapy (n=8), or no treatment at all (n=10). Yet symptoms resolved and no further C. difficile testing was requested for 13 of 18 patients for 6-8 months after hospital discharge. Only one patient had a subsequent cytotoxin positive stool submitted 22 days after the study sample was tested. Enhanced sensitivity and rapid turnaround time make the BD GeneOhm Cdiff assay an important advance in the diagnosis of toxigenic C. difficile infection. The BD GeneOhm Cdiff assay is significantly more sensitive than a commonly-used ELISA toxin assay and has a sensitivity and specificity comparable to the two-step method. Its turnaround time is similar to ELISA toxin assays and more rapid than the two-step method. Disadvantages to implementation of BD GeneOhm Cdiff assay include increased cost and potential treatment of asymptomatic carriers and mild, self-resolving disease.

Clostridium difficile is a leading cause of nosocomial infection in North America and a considerable challenge to healthcare professionals in hospitals and nursing homes. The Gram-positive bacterium produces two exotoxins, toxin A (TcdA) and toxin B (TcdB), which are the major virulence factors responsible for C. difficile-associated disease (CDAD) and are targets for CDAD therapy. In this work, recombinant single-domain antibody fragments (VHHs) which target the cell receptor binding domains of TcdA or TcdB were isolated from an immune, llama phage display library and characterized. Four VHHs (A4.2, A5.1, A20.1, and A26.8) were potent neutralizers of the cytopathic effects of TcdA in an in vitro assay and the neutralizing potency was enhanced when VHHs were administered in combinations. Epitope mapping experiments revealed that some synergistic combinations consisted of VHHs recognizing overlapping epitopes, an indication that factors other than mere epitope blocking are responsible for the increased neutralization. Binding assays revealed TcdA-specific VHHs neutralized TcdA by binding to sites other than the carbohydrate binding pocket of the toxin. The TcdB-specific VHHs failed to neutralize TcdB, as did a panel of human VL antibodies isolated from a synthetic library. To enhance the stability of the C. difficile TcdA-specific VHHs for oral therapeutic applications, the VHHs were expressed with an additional disulfide bond by introducing Ala/Gly54Cys and Ile78Cys mutations. The mutant VHHs were found to be well expressed, were non-aggregating monomers, retained low nM affinity for TcdA, and were capable of in vitro TcdA neutralization. Digestion of the VHHs with the major gastrointestinal proteases, at biologically relevant concentrations, revealed a significant increase in pepsin resistance for all mutants and an increase in chymotrypsin resistance for the majority of mutants without compromising inherent VHH trypsin resistance. Collectively, the second disulfide not only increased VHH thermal stability at neutral pH, as previously shown, but also represents a generic strategy to increase VHH stability at low pH and impart protease resistance. These are all desirable characteristics for the design of protein-based oral therapeutics. In conclusion, llama VHHs represent a class of novel, non-antibiotic inhibitors of infectious disease virulence factors such as C. difficile toxins.

Thesis (M.S.)--University of South Florida, 2007. / Title from PDF of title page. Document formatted into pages; contains 89 pages. Includes bibliographical references.

SANTOS, A. A. Q. A. Papel do receptor P2x7 no efeito da toxina a do Clostridium difficile em modelo de alça ileal em camundongos. 2017. 100 f. Tese (Doutorado em Ciências Médicas) - Faculdade de Medicina, Universidade Federal do Ceará, Fortaleza, 2017. / Submitted by Erika Fernandes (erikaleitefernandes@gmail.com) on 2017-01-30T16:00:58Z No. of bitstreams: 1 2017_tese_aaqasantos.pdf: 2658637 bytes, checksum: 8c134a5e88286c7d898fc1d1a091d893 (MD5) / Approved for entry into archive by Erika Fernandes (erikaleitefernandes@gmail.com) on 2017-01-30T16:01:09Z (GMT) No. of bitstreams: 1 2017_tese_aaqasantos.pdf: 2658637 bytes, checksum: 8c134a5e88286c7d898fc1d1a091d893 (MD5) / Made available in DSpace on 2017-01-30T16:01:09Z (GMT). No. of bitstreams: 1 2017_tese_aaqasantos.pdf: 2658637 bytes, checksum: 8c134a5e88286c7d898fc1d1a091d893 (MD5) Previous issue date: 2017-01-16 / Clostridium difficile (C. difficile) is the major cause of colitis associated with the use of antibiotics, with significant morbidity and mortality. The enteric nervous system (ENS) is located in the gastrointestinal tract (GIT) and has an important function of regulating the digestive system. The P2X7 receptor participates in the regulation of cellular permeability, cytokine release and apoptosis, processes that are involved in the pathogenesis of C. difficile induced disease.The aim of this study was to analyze the role of the P2X7 receptor in enteritis induced by C. difficile toxin A (TcdA). The mouse ileal loop was injected with saline (Sodium Chloride 0.9% - Control) or TcdA (50 μg / loop) in mice previously treated with the inhibitors: BBG (50 mg/kg) or A438079 (10 μM) i.p.injection 1 h prior to TcdA (TcdA + BBG; TcdA + A438079) or saline injection. The animals were euthanized after 4h latter and the ileal loop collected for the following analyzes: immunofluorescence for ChAT, NOS, P2X7 and calretinin in membrane preparations, immunohistochemistry for GFAP, HuC / D, S-100β, TUNEL and PCR for P2X7. The results demonstrated that TcdA significantly reduced myenteric plexus neurons in 66.42% when compared to the control, while in the membrane preparations a significant reduction of the immunoreactive neurons to ChAT was observed in 23.21% and Calretinin 28.17%. However, there was a 21.23% increase in labeling for the P2X7 receptor over the control. In the ileal tissue there was an increase of 411.95% and 22.40% respectively in the P2X7 positive area and in the gene expression. In relation to the glial markers, an increase of 125.71% and 144.30% was observed for GFAP and S-100β, respectively. In animals treated with inhibitors P2X7; BBG and A438079, as observed an improvement in histological parameters . In addition , a significant reduction in the number of apoptotic cells decrease in GFAP and S-100β-labeled cells, and cytokines ( TNF-α, IL-1β, IL -6 and IL-8) levels, compared to the control. This was associated with a significant increase in the HuC/D positive area . From these results, it is concluded that TcdA promotes changes in the enteric nervous system, increasing neuronal death and activating glial cells, and that the P2X7 receptor plays an important role in the changes induced by C. difficile TcdA. / O Clostridium difficile (C. difficile) é a maior causa de colite associada ao uso de antibióticos, com significante morbidade e mortalidade. O sistema nervoso entérico (SNE) está localizado no trato gastrointestinal (TGI) e possui importante função de regulação do sistema digestório. O receptor P2X7 presente nas células gliais entéricas (CGE) e no SNE participa na regulação da permeabilidade celular, liberação de citocinas e apoptose, processos que estam envolvidos na patogênese do C. difficile. O objetivo deste estudo foi analisar o papel do receptor P2X7 na enterite induzida pela toxina A (TcdA) do C. difficile. A alça ileal dos camundongos foi injetada com salina (Cloreto de Sódio 0,9%- Controle) ou TcdA (50 μg/alça) em camundongos previamente tratados com os inibidores: BBG (50 mg/kg) ou A438079 (10 μM) via intraperitoneal 1h antes da injeção da TcdA ou salina. Os animais foram eutanaziados após 4h da injeção e a alça ileal coletada para as seguintes analises: imunofluorescência para ChAT, NOS, P2X7 e calretinina nos preparados de membrana, imunohistoquímica para GFAP, HuC/D, S-100β, TUNEL e PCR para P2X7. Os resultados demonstraram que a TcdA reduziu significativamente em 66,42% os neurônios do plexo mioentérico quando comparado ao controle, enquanto nos preparados de membrana observou-se uma redução significativa dos neurônios imunorreativos a ChAT em 23,21% e Calretinina 28,17%, contudo, houve um aumento de 21,23% da marcação para o receptor P2X7 em relação ao controle. No tecido ileal verificou-se um aumento de 411,95% e 22,40% respectivamente na área positiva para P2X7 e na expressão gênica. Em relação aos marcadores gliais, observou-se um aumento de 125,71% e 144,30% para GFAP e S-100β, respectivamente. Nos animais tratados com os inibidores de P2X7: BBG e o A438079 observou-se uma melhora dos parâmetros histológicos, uma redução significativa do número de células apoptóticas, das células marcadas para GFAP e S-100β, das citocinas: TNF-α, IL-1β, IL-6 e IL-8 com relação ao controle e um aumento significativo na área positiva para HuC/D em relação a TcdA. A partir desses resultados, concluiu-se que a TcdA promoveu alterações no sistema nervoso entérico, aumentando a morte neuronal e ativando as células da glia, e que o receptor P2X7 tem participação importante nas alterações induzidas pela TcdA do C. difficile.

nÃo hà / Diarreia nosocomial (DN) à uma infecÃÃo relacionada à assistÃncia à saÃde (IRAS) com incidÃncia e severidade crescentes. PropÃe-se determinar a incidÃncia da DN, os fatores de risco e a incidÃncia da doenÃa associada a Clostridium difficile (C. difficile). Para isso, um estudo caso â controle, pareando pacientes por idade, sexo, perÃodo de admissÃo, clÃnica e diagnÃstico, foi conduzido, de 06/ fev/12 a 05/fev/13, no Hospital UniversitÃrio da UFC. Casos &#822; pacientes com DN e Controles &#822; pacientes sem DN. Definiu-se DN como fezes lÃquidas, trÃs ou mais vezes em 24 horas, com duraÃÃo superior a 12 horas, sem outras causas inflamatÃrias ou procedimentos diagnÃsticos. DN foi detectada mediante busca ativa, visitando-se os pacientes das Unidades de Hematologia, Transplante HepÃtico e Renal. O teste ELISA TOX A/B II foi utilizado para detectar as toxinas A e/ou B e diagnosticar doenÃa associada ao C. difficile. Demais IRAS foram investigadas por intermÃdio de fichas de notificaÃÃo de infecÃÃo hospitalar (IH). O Ãndice geral de IH foi de 7,17%. A incidÃncia da DN nas enfermarias de Hematologia, Transplante HepÃtico e Renal foi 4,80% (44/925) e da DN associada ao C. difficile 0,12% (01/925). Detectaram-se toxinas A/B de C. difficile em caso [1/43 (2,32%)] e controles [3/72 (4,17%)]. DN foi significantemente associada ao uso prÃvio > 6 antimicrobianos por paciente, alÃm do uso prÃvio de ciprofloxacina, metronidazol, polimixina B e dieta enteral (p&#8804; 0,05). Pacientes com DN permaneceram mais tempo internados, tiveram mais vÃmitos, cÃlicas e febre, verificando-se alta significÃncia estatÃstica (p&#8804; 0,05). Outras IRAS identificadas, nos casos e controles, foi infeÃÃo do trato urinÃrio 54% (15/28), seguida da corrente sanguÃnea 32% (8/28), do sÃtio cirÃrgico 11% (3/28) e de infecÃÃo de partes moles 4% (1/28). DN impÃe riscos aos pacientes jà debilitados. Os dados demonstram a presenÃa endÃmica do C. difficile. A atualizaÃÃo da epidemiologia local orienta medidas de controle da IH, como uso judicioso de antibiÃticos, cautelas com a dieta enteral e precauÃÃes de contato, para os pacientes com diarreia nosocomial. / Nosocomial diarrhea (ND) is a healthcare - associated infections (HAI) with increasing incidence and severity. It is proposed to determine the incidence of ND, the associated risk factors and the incidence of disease associated to Clostridium difficile (C. difficile). For this, a case - control study, pairing patients by age, sex, length of admission, and clinical diagnosis was conducted 06 / Feb/12 to 05/Fev/13 in the University Hospital of the UFC. Cases: patients with DN and controls: patients without ND. Nosocomial diarrhea is defined as watery stools, three or more times within 24 hours, over 12 hours without further diagnostic procedures or inflammatory causes. ND was detected by active surveillance, visiting the patients of Hematology, Liver and Renal Transplant. DN was defined as loose stools, 3 or more times in 24 hours, with duration longer than 12 hours, without other inflammatory causes or diagnostic procedures. The ELISA TOX A / B II test was used to detect toxin A and/or B and to diagnose C. difficile associated disease. Others HAI were investigated by the notification records of nosocomial infection (NI). The overall rate of Nosocomial infection was 7.17 %. The incidence of DN in the wards of Hematology, Liver and Renal Transplant was 4.80% (44/925) and C. difficile associated with DN was 0.12 % (01/925). Toxins A/B were detected in the case of C. difficile [1/43 (2.32%)] and controls [3/72 (4.17%)]. DN was significantly associated with previous use > 6 antimicrobials per patient, beyond the prior use of ciprofloxacin, metronidazole, polymyxin B and enteral feeding (p &#8804; 0.05). Patients with ND remained in hospital longer, had more vomiting, cramps and fever, verifying high statistical significance (p &#8804; 0.05). Other identified HAI were mainly urinary infection 54% (15/28), followed by bacterial bloodstream infection 32% (8/28), surgical site infection 11% (3/28) and soft tissue infection 4% (1/28). ND entails risks to the already debilitated patients. The data demonstrate the presence of endemic C. difficile. The updated of the local epidemiology guide control measures NI, such as judicious use of antibiotics, enteral feeding precautions and contact precautions for patients with nosocomial diarrhea.