CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Clostridium difficile à um bacilo Gram positivo, anaerÃbio estrito, formador de esporos e produtor de toxinas. Atualmente, representa a principal causa de diarreia hospitalar associada ao uso de antibiÃticos. Pacientes oncolÃgicos sÃo um dos principais grupos de risco para infecÃÃo por C. difficile (CDI), visto que o uso de agentes quimioterÃpicos pode alterar a mucosa intestinal. AlÃm disso, estes pacientes normalmente sÃo imunodeprimidos e frequentemente utilizam antibiÃticos de largo espectro. Tendo em vista a patogenicidade do C. difficile e a importÃncia da doenÃa induzida por essa bactÃria em ambiente hospitalar este estudo visou determinar a incidÃncia e caracterizaÃÃo fenotÃpica e genotÃpica de cepas de C. difficile isoladas de pacientes oncolÃgicos internados do Hospital Haroldo JuaÃaba, Fortaleza, CearÃ. Durante o perÃodo de 12 meses (maio/2013 a maio/2014) foram coletadas 41 amostras de fezes diarreicas. Toxinas A e/ou B foram detectadas a partir das fezes por meio de um kit de detecÃÃo comercial ELISA. Em seguida, as amostras foram cultivadas em Agar Cicloserina, Cefoxitina, Frutose (CCFA) e incubadas em anaerobiose. As cepas isoladas foram processadas e realizadas identificaÃÃo fenotÃpica e anÃlise de detecÃÃo dos genes das toxinas e do fragmento do gene tpi (identificaÃÃo definitiva) por PCR convencional. A sensibilidade das cepas isoladas a 12 antimicrobianos foi determinada por meio de E-test. TambÃm foi realizado a genotipagem das cepas por meio da anÃlise molecular PFGE. 46,3% (19/41) das amostras foram positivas para presenÃa das toxinas A/B por ELISA e/ou cultura do C. difficile. Dessas amostras, foram isolados C. difficile de trÃs amostras (15,8% - 3/19). Em todos os isolados foram detectados os genes tpi, tcdA e tcdB. O domÃnio de ligaÃÃo da toxina binÃria (cdtB) nÃo foi detectado assim como tambÃm nÃo foram observadas deleÃÃes no gene tcdC nos isolados. Todas as cepas apresentaram o mesmo genÃtipo, NAP4. Com relaÃÃo à sensibilidade das cepas aos antimicrobianos foi verificado resistÃncia a dois ou mais antimicrobianos (azitromicina, tetraciclina, ciprofloxacina, levofloxacina, ceftriaxona e cefotaxima). 57,9% (11/19) faziam uso de antibiÃticos e quimioterÃpicos. Este trabalho descreveu a incidÃncia de CDI em pacientes oncolÃgicos, e evidenciou pela primeira vez a presenÃa de C.difficile em casos associados a comunidade (CA-CDI) nesses pacientes no Brasil, ressaltando a importÃncia do estudo dessa bactÃria para a compreensÃo da situaÃÃo epidemiolÃgica dessa infecÃÃo e de sua dispersÃo entre unidades hospitalares brasileiras. / Clostridium difficile is a strictly anaerobic, spore-forming, toxin-producing Gram positive bacillus. Currently, it is the main cause of nosocomial diarrhea associated with antibiotic use. Cancer patients are a major risk group for C. difficile infection (CDI), since the use of chemotherapeutic agents can alter the intestinal mucosa. Furthermore, these patients are often immunosuppressed and often use broad spectrum antibiotics. Considering the pathogenicity of C. difficile and the importance of this infection in hospitalized patients, this study aimed to determine the incidence and the phenotypical and genotypical characterization of strains of C. difficile isolated from cancer patients at Haroldo JuaÃaba Hospital, Fortaleza, CearÃ. During the 12 month period (May/2013 to May/2014) 41 diarrheic fecal samples were collected. Toxins A/B were detected from feces through a commercial ELISA detection kit. Then, the samples were cultivated on cefoxitine-cycloserine-frutose agar (CCFA) and incubated anaerobically. Isolates were submitted to several analyses, including phenotypical identification, detection of toxin genes and of a fragment of the tpi gene (definitive identification) by conventional PCR. The susceptibility of the strains to 12 antimicrobial agents was determined by E-test. Genotyping of the strains was also performed through molecular PFGE analysis. Out of 41 samples, 46.3% (19/41) were positive for either one or both of the performed tests: detection of toxin A/B and/or culture of C. difficile. C. difficile was recovered from three samples (15.8% - 3/19). The tpi, tcdA and tcdB genes were detected in all of the isolates. The binding domain of the binary toxin (cdtB) was not detected as well as no deletions were observed in the tcdC gene of the analysed isolates. All strains belonged to the same genotype, NAP4. Regarding the antimicrobial susceptibility of the strains, resistance to two or more antibiotics (azithromycin, tetracycline, ciprofloxacin, levofloxacin, ceftriaxone and cefotaxime) was observed. Out of the 19 positive patients, 57.9% (11/19) were using antibiotics and under chemotherapy. This paper describes the incidence of CDI in patients with cancer, and shows for the first time the detection of community-associated Clostridium difficile infection (CA-CDI) in those patients in Brazil, highlighting the importance of studying this bacterium for understanding the epidemiological situation of this infection and its spread among Brazilian hospitals.

Protonpumpshämmare är en grupp läkemedel som globalt sett tillhör de mest använda läkemedlen. De används vid t.ex. magsårssjukdomar och hämmar utsöndring av saltsyra i magsäcken vilket leder till att pH ökar. Clostridium difficile är en grampositiv stav- och sporbildande bakterie som vid antibiotikabehandling kan orsaka svåra diarréer. Det misstänks att protonpumpshämmare (PPI) kan orsaka överväxt av Clostridium difficile. Mekanismen bakom detta är fortfarande oklar men den vegetativa formens och sporernas överlevnad i magsäcken verkar underlättas vid minskning av magsyran, d.v.s. medicinering med PPI. Syftet med denna studie var att undersöka om medicinering med syrahämmande läkemedel, protonpumpshämmare, ökar risken för utveckling av Clostridium difficile infektion. Metoden som användes var en litteraturstudie av fall-kontroll-och kohortstudier samt metaanalyser från databasen Pubmed. Resultaten från samtliga studier, förutom en fall-kontrollstudie, påvisade ett statistiskt signifikant ökat odds att drabbas av Clostridium difficile infektion och även återkommande infektion vid PPI-användning. Slutsatsen är att det krävs kliniska prövningar för att styrka denna observation, dock är det sannolikt omöjligt att genomföra sådana dels p.g.a. behovet av ett stort antal försökspersoner samt kostnaden. De granskade studierna är inga kliniska prövningar med högt bevisvärde, utan av typen fall-kontroll- och kohort-studier. Dock påvisas statistiskt signifikant koppling mellan behandlingen med PPI och utveckling av Clostridium difficile infektion i samma riktning även i många andra studier. Åtgärder bör vidtas för att minska överanvändning av PPI och för att använda dem endast när det välmotiverat. Läkare och befolkningen i allmänhet bör också uppmärksammas på denna allvarliga komplikation som är kopplad till medicinering med protonpumpshämmare.

Clostridium difficile infection (CDI) is the most common cause of hospital-acquired infectious diarrhea in the United States. Although C. difficile is widely-recognized as a pathogen among hospitalized populations, CDI has emerged in the community setting but is under-diagnosed. This study sought to increase knowledge about the incidence of, risk factors for, and outcomes associated with community-associated CDI (CA-CDI). A retrospective nested case-control study was conducted using insurance claims data from the Wellmark Data Repository for the time period between January 1, 2003 and December 31, 2007. Persons with CDI were identified and were classified as community-associated CDI and hospital-acquired CDI. During this time, 304 cases of CA-CDI and 338 cases of HA-CDI were identified. Within this population, the incidence rate for CA-CDI was 11.16 cases per 100,000 person-years, whereas the incidence rate for HA-CDI was 12.41 cases per 100,000 person-years. Conditional logistic regression was utilized to determine the risk for CA-CDI related to pharmacologic exposures, comorbidity, demographic characteristics, and healthcare utilization. Prior to controlling for other risk factors and covariates; being over the age of 50 years, gender, history of hospitalization, number of outpatient physician visits, antimicrobial use, gastric acid suppressant use, underlying comorbidity, and diagnosis of gastrointestinal disease (including IBD, diverticular disease, GERD) were associated with the development of CA-CDI. However, after adjustment for all covariates, increased risk for CA-CDI within this population was consistently associated with antimicrobial use, being between the age of 19 and 74 years, and diagnosis of inflammatory bowel disease. Gastric acid suppressant use was a risk factor in a number of models, although this association was not consistent. Furthermore, persons who last received antimicrobials in the previous 150 days and persons who received a greater number of different antimicrobial agents were at increased risk for CA-CDI. Antimicrobial use was the primary risk factor for CA-CDI, although 27% of cases did not have prior exposure to antimicrobials. In fact, 17% of CA-CDI cases did not have any of the traditional risk factors for CDI (i.e., no antimicrobial or gastric acid suppressant exposure, no underlying illness, and no history of hospitalization). Furthermore, none of the CA-CDI cases underwent surgical procedures attributable to CA-CDI, although approximately 25% of CA-CDI cases were hospitalized with a diagnosis of CDI. This research demonstrates that CDI is occurring in the community setting and in populations that were previously not considered to be at risk. In this study, the risk factors for CA-CDI were similar to those identified in hospitalized populations, although it was not uncommon for persons to develop CA-CDI without any of these risk factors. Furthermore, the characteristics of persons with CA-CDI and the outcomes in this group were different than those previously reported among hospital-acquired CDI cases. Collectively, this study provides valuable knowledge about the epidemiology of CA-CDI and serves as a foundation for future research.

Objetivo: Evaluar si la infección por Clostridioides difficile se asocia a una mayor frecuencia de complicaciones (muertes, colectomías, obstrucción intestinal, visitas a emergencia, número de exacerbaciones) en pacientes de 18 años a más con enfermedad inflamatoria intestinal (Colitis ulcerativa y/o Enfermedad de Crohn) en comparación a los no infectados. Diseño: Se realizará una Revisión sistemática de la literatura científica disponible que evalúen si la infección por Clostridioides difficile se asocia a una mayor frecuencia de complicaciones (muertes, colectomías, obstrucción intestinal, visitas a emergencia, número de exacerbaciones) en pacientes de 18 años a más con enfermedad inflamatoria intestinal (Colitis ulcerativa y/o Enfermedad de Crohn) en comparación a los no infectados. Para esto se seguirá el método Cochrane y la lista de chequeo PRISMA para la realización de revisiones sistemáticas.

Loop-mediated isothermal DNA amplification (LAMP) are currently used as standalone diagnostic test for C. difficile infection (CDI). We assessed the diagnostic accuracy of LAMP for the diagnosis of CDI. We searched 5 databases to identify studies that compared LAMP with culture cytotoxicity neutralization assay or anaerobic toxigenic culture (TC) of C. difficile. We used the random-effects model to calculate pooled sensitivities, specificities, diagnostic odds ratios and their 95% confidence intervals (CIs). The search of the databases yielded 16 studies (6,979 samples) that met inclusion criteria. When TC was used as the gold standard (6,572 samples), bivariate analysis yielded a mean sensitivity of 0.95 (95%CI, 0.93-0.97; I2 = 67.4) and a mean specificity of 0.99 (95%CI, 0.96-1.00; I2 = 97.0). LAMP is a useful diagnostic tool with high sensitivity and specificity for detecting CDI. The results should however be interpreted only in the presence of clinical suspicion and symptoms of CDI. / Revisión por pares

<p><em>Clostridioides difficileinfection</em>  (CDI)  is  considered  as  an  urgent  threat  to  the  publicby  CDC, 2019.It  causes  life-threatening  diarrhea  and pseudomembranous colitis,mostly  in those  taking antibiotics or at the end of their antibiotic course.It is also notifiedas hospital-associated pathogensbecause one-third  of  the CDIhas occurredinthe health  care  center. Norepinephrine  (NE)  is  a stress-associated  neuroendocrine  hormone  released  upon  sympathetic  stimulation  to  mediate stress.Gut walls are highly innervated by the sympathetic nervous system. During stress, elevated level  of  NE  released  in  the  GI  tractcould  influence  bacterial  overgrowth  &  translocation.  It  isalready known  for  its  role  in  modulating  the  behavior  of  several  bacterial  pathogens  suchas Staphylococcus, Escherichia coli, Salmonella, and Vibrio cholera. This study aims to evaluate the effect of NE treatment on the growth and virulence of C. difficile.Here, we studied the effect of NE  on  six  different C.  difficilestrains  isolated  from humans. To  understandthe  influence  on growth, bacterial culture was treated (+/-)NE (5μM & 50 μM)during their log phase and recorded the density of the cell each time period for constructing the growth curve. In addition, after NE treatment, bacterial cells were taken for further analysis. For investigating the impact of NE on the virulence  genes  expression, a qPCR  reaction  was  performed  along  with -RT  / noRT  control reactions  for assessingthe  RNA  sample  free  from  genomic  DNA  contamination.  In the case of growth,higher growth was observed in VPI 10463at 6 hourtime pointonly,and in strain,NR 49277 significantly stimulated after 6 hoursand continued till 8 hours after treatmentwith50μM NE. In strain NR 49282, decreasedgrowth was observed at7-hourtime pointsafter 50 μM NEtreatment.But, there was no difference in cell density between control &  5μM NE treated bacterial culture in all strains.</p> <p>Toxingenes(tcdA&tcdB)and flagellin gene(fliC),were upregulated in NR 49290, NR 49277 & VPI 10463strains in both concentrations of NE and down-regulated in NR 49282.In strain NR 32888, toxin genes were downregulated while treated with 5μM NEbut upregulated after 50μM NEtreatment, though fliC was downregulated in both concentrations. In strain NR 32891,  tcdAwas downregulated,but tcdB& fliCwere upregulatedafter NE treatmentin both concentrations. Increased expression in pilin gene,pilA1in strain NR 49277, NR 49290, VPI 10463& NR 32891 in both concentrationswas observed.  In addition, pilA3in NR 49277, VPI 10463& NR 32891 and PilA5in  NR  49277  &  NR  49290  showed an upregulation pattern while  treated  with  both concentrations. Modulating this response, it is possible to reduce the pathogenicity of C. difficileduring medical care & antibiotic use.</p> <p><br></p>

Background: Clostridium difficile is the most common cause of healthcare-associated infections in the United States, and has been associated with adverse outcomes in the acute care setting. However, little is known regarding the burden or impact of C. difficile infection (CDI) in long-term acute care hospitals (LTACHs). Methods: A retrospective matched cohort study was performed among patients at an urban, university-affiliated LTACH between July 2008 and October 2015. The incidence rate of LTACH-onset CDI was assessed and patient characteristics associated with adverse outcomes examined. Patients with CDI were matched to concurrently hospitalized LTACH patients without a diagnosis of CDI. A multivariable model using logistic regression was developed to determine characteristics associated with a composite primary outcome of either 30-day readmission to an acute care hospital or mortality. Subgroup analyses were performed for patients with a diagnosis of severe CDI. Results: The overall incidence of CDI was 21.4 cases per 10,000 patient-days. Patients with CDI had a mean age (±SD) of 70 ±14 years and a mean admission Charlson Comorbidity Index (CCI) of 4 ±2. Median (IQR) time between admission and diagnosis of CDI was 16 days (range: 9-23 days). In the final multivariable model, CDI was not a significant risk factor for the primary outcome (OR, 1.06 [95% confidence interval {CI}, 0.53-2.10]). Congestive heart failure (OR, 2.27 [95% CI, 1.15-4.57]), albumin level (OR, 0.44 [95% CI, 0.22-0.79]), and immunosuppression (OR, 2.94 [95% CI, 1.06-8.39]) were independent risk factors for the primary outcome. On subgroup analysis, severe CDI and CCI were significant risk factors for the primary outcome in bivariable analysis (OR, 2.91 [95% CI 1.03-8.20] and OR, 1.36 [95% CI 1.06-1.80], respectively). Only CCI remained significant in the multivariable model (OR, 1.32 [95% CI 1.02-1.75]). Conclusions: LTACH-onset CDI was found to have a relatively high incidence in an urban, university affiliated LTACH. CDI was not a significant risk factor for the composite outcome of 30-day readmission or mortality. Future research should focus on infection prevention and antibiotic stewardship measures to decrease CDI specifically in the LTACH setting. / Epidemiology

Over the last 20 years, the incidence of Clostridium difficile infection (CDI) has increased dramatically, making it one of the most common healthcare-associated infections. This has been linked to the emergence of hypervirulent C. difficile strains. Currently, cell cytotoxicity assay (CTA) and toxigenic culture are the gold-standard methods for CDI diagnosis. However, they are time-consuming and labour-intensive. Other methods, like enzyme immunoassays (EIAs) and nucleic acid amplification-based tests (NAATs), allow for rapid testing but have poor sensitivity and/or specificity. Additionally, most of these methods target toxins or their associated genes and are unable to discriminate between epidemic and non-epidemic strains. The work described in this dissertation aims to develop easy-to-use and reliable biosensors for C. difficile, with a particular focus on epidemic strains of C. difficile. The development of the in vitro selection technique has allowed for the discovery of a big array of functional DNA, with excellent ability in both target recognition and enzymatic catalysis. My key interest is to employ functional DNA molecules as target recognition elements to develop colorimetric biosensors for C. difficile detection. The first research project aimed to develop a colorimetric detection platform that can be coupled with functional DNA molecules to achieve hypersensitive detection of different targets. This test should be easy-to-use, have broad target applicability and not require expensive equipment. To do so, a colorimetric biosensing platform was created, which takes advantage of the signal amplification ability of rolling circle amplification (RCA) and the simplicity of the classic litmus test. In the presence of the target of interest, RCA will be triggered, and the biotinylated RCA products can hybridize a number of the urease-labelled single-stranded DNA and immobilize urease onto magnetic beads through streptavidin-biotin interactions. The urease can then be used to hydrolyze urea, resulting in significant pH elevation, that can be detected easily using a litmus test. To prove the concept, we have demonstrated that this platform can be employed to visually detect thrombin and platelet-derived growth factor (PDGF) with high sensitivity, by coupling it with an anti-thrombin aptamer and an anti-PDGF aptamer, respectively. We have also shown that the biosensing platform can be incorporated into simple paper-based devices. The second project focuses on the development of a colorimetric DNA detection method for epidemic strains of C. difficile that utilizes both the polymerase chain reaction (PCR) and the litmus test. The strategy makes use of a modified set of primers for PCR to facilitate ensuing manipulations of resultant DNA amplicons: their tagging with urease and immobilization onto magnetic beads. The amplicon/urease-laden beads are then used to hydrolyze urea, resulting in an increase of pH that can be conveniently reported by a pH-sensitive dye. We have successfully applied this strategy for the detection of two hypervirulent strains of C. difficile, which are responsible for the recent increase in the global incidence and severity of C. difficile infections. Furthermore, the viability of this test for diagnostic applications is demonstrated using clinically validated stool samples from C. difficile infected patients. The goal of the third project was to isolate RNA-cleaving fluorogenic aptazymes (RFAs) targeting an epidemic strain of C. difficile. Four classes of RFA probes were derived using in vitro selection approach where a random-sequence DNA library was reacted with a crude extracellular mixture (CEM) derived from the epidemic C. difficile strain BI/027/NAP1, coupled with a subtractive selection strategy to eliminate cross-reactivities to unintended C. difficile strains and other bacterial species. The isolated RFDs can be used together to generate specific cleavage patterns for strain-specific identification of C. difficile. Lastly, the final project was to characterize a novel RFA probe (RFA13-1) that was isolated unintentionally using in vitro selection. By using CEM prepared from C. difficile glycerol stock contaminated by Klebsiella aerogenes as the positive target for in vitro selection, we isolated a remarkably active RFA probe, RFA13-1, targeting K. aerogenes. Further studies demonstrated that RFA13-1 could be activated by CEM prepared from several bacteria from the Enterobacteriaceae family. Moreover, the molecular target of RFA13-1 has been identified, which is ribonuclease I. RFA13-1 showed high sensitivity and specificity towards RNase I and could be employed as a tool to study RNase I functions and to detect RNase I or RNase I-containing bacteria. In summary, I have investigated novel strategies for building a biosensor that is capable of discriminately detecting epidemic strains of C. difficile. I hope that my work can take us one step closer towards the development of easy-to-use and reliable biosensors that can be used in the clinical diagnosis of CDI. / Thesis / Doctor of Philosophy (PhD)

Clostridium difficile is a major nosocomial pathogen responsible for causing pseudomembranous colitis. It is estimated that 25% of antibiotic-associated diarrhea is due to C. difficile. These diseases result from intestinal tissue damage caused by two of the largest known bacterial toxins, A and B. Molecular studies of the C. difficile toxins have identified a 19.6 kb toxigenic element that contains both toxin genes flanked by three small open reading frames (ORFs). The focus of this study is to elucidate the function of the ORF, designated txeR, which is located at the beginning of the toxigenic element. The deduced amino acid sequence of txeR predicts a 22-kDa protein that contains a helix-turn-helix motif characteristic of DNA binding regulatory proteins. To determine if the protein TxeR regulates expression from the toxA, toxB, and txeR promoters, gene fusions were constructed that contained the various promoter regions and a reporter gene. The immunodominant region of toxin A located at the carboxy-terminus, termed the repeating units (ARU), was selected as the reporter gene. Expression studies were performed in Escherichia coli host strains. Levels of ARU expression were measured by enzyme-linked immunosorbent assay using an ARU-specific monoclonal antibody. Expression levels of ARU from the toxin B promoter region with TxeR supplied on the same plasmid (in cis) or on a different plasmid (in trans) were determined. In cis, ARU levels were 50-fold higher than strains without txeR. In trans, expression of ARU from the toxin B promoter region increased over 800-fold. When TxeR was supplied in trans to a toxin A promoter region-ARU fusion, expression levels of ARU increased over 500-fold. To test for autoregulation, TxeR was supplied in trans to the txeR promoter region fused to ARU. The effect was an increase of ARU expression up to 20-fold over background. These results suggest that TxeR is a trans-acting regulator that stimulates expression of the C. difficile toxins and is subjected to autoregulation. / Master of Science

Clostridium difficile causes pseudomenbranous colitis (PMC) and diarrhea in humans. Toxigenic strains of C. difficile produce two toxins. Toxin A is an enterotoxin and cytotoxin, and toxin B is a potent cytotoxin. The gene encoding toxin A has been sequenced and was shown to possess a 2.5 kb region, containing 38 similar repeating amino acid sequences, at the 3' -end of the gene. This region of the toxin A gene codes for the carbohydrate-binding portion of the toxin. The monoclonal antibody PCG-4 (MAb) binds to this portion of toxin A and neutralizes its enterotoxic activity. In addition, this monoclonal antibody has been shown to immunoprecipitate toxin A, suggesting that the MAb PCG-4 is binding to two or more similar epitopes on the toxin. The goal of this research project was to identify the neutralizing epitopes recognized by the MAb PCG-4 on the surface of the toxin A. To map the epitopes bound by the MAb PCG-4, a series of overlapping deletion clones were constructed from a 4.7 kb fragment from the 3'-end of the toxin A gene. The recombinant polypeptides expressed by these clones were tested for reactivity with the MAb PCG-4. By comparing the overlapping polypeptides, defined as either PCG-4 reactive or nonreactive, I localized the PCG-4 epitope to a 44-amino acid sequence situated between the amino acid residues 2098-2141 of toxin A A similarity search of the toxin with the 44-amino acid sequence containing the PCG-4 epitope revealed the presence of two other possible PCG-4 epitopes located between the amino acid residues 2355-2398 and 2459-2502. However, subsequent cloning experiments showed that only the region located between the amino acid residues 2355-2398 contained a PCG-4 reactive epitope. The identification of two similar epitopes within the toxin's structure explains how this monoclonal antibody is able to immunoprecipitate toxin A in the absence of subunits. Furthermore, I found that small recombinant polypeptides, containing the PCG-4 epitope lost reactivity with this monoclonal antibody following denaturation, suggesting that the epitopes recognized by this monoclonal antibody are conformationally dependent. / Master of Science