Status and challenge of vaccine research for Clostridiodes difficile in clinical trials / 中国生物制品学杂志

@#Clostridiodes difficile(C.difficile)is the most common causative agent of antibiotic-associated diarrhea(ADD)in the world. In recent years,with the emergence of highly resistant and virulent strains,the outbreaks of C.difficile infection have occurred around the world. The incidence,recurrence and mortality of C.difficile infection are on the rise worldwide,and bring great challenges to clinical treatment. Pathogenic strains mainly produce two homologous glycosylation toxins A and B,which can cause symptoms ranging from diarrhea to highly lethal toxic megacolon. In view of the malignant consequences caused by C.difficile infection,disease prevention is still an important way worth exploring. Until now there is no approved vaccine against C.difficile. Therefore,this review assessed the status and challenge of clinical trials of vaccine research for C.difficile.

Clostridioides difficile: achados epidemiológicos em animais domésticos em um Hospital Veterinário Universitário em ­ Teresina, PI / Clostridioides difficile: epidemiological findings in domestic animals in a University Veterinary Hospital in Teresina, PI

O objetivo deste estudo foi analisar a prevalência de Clostridioides difficile e suas toxinas (A/B) nas fezes de animais domésticos de um Hospital Veterinário Universitário de Teresina - PI. A detecção de C. difficile e suas toxinas foi realizada por meio de um ensaio imunoenzimático, denominado C. Diff Quik Chek Complete® (TECHLAB), capaz de detectar antígeno Glutamato Desidrogenase (GDH) e as toxinas A/B produzidas pelo bacilo, realizado em amostras fecais de cães (C. lupus) e e gatos (Felis catus) coletadas entre agosto de 2019 a setembro de 2020. Um total de 54 amostras fecais foram analisadas, das quais 16 foram positivas para C. difficile (29,63%). 68,75% (11/16) pertenciam a caninos, enquanto 31,25% (5/16) a felinos. Amostras diarreicas e não diarreicas foram utilizadas para o estudo e uma maior prevalência do bacilo pôde ser identificada em amostras diarreicas (33%). Nenhuma das amostras apresentou toxinas do patógeno. Os achados deste estudo evidenciam que C.difficile está presente no estado do Piauí. Foi possível identificá-lo em todas as espécies e em amostras diarreicas ou não, demonstrando que essa infecção pode se manifestar de formasintomática e assintomática, levantando a possibilidade de infecção cruzada entre o animal e seu tutor.

The aim of this study was to analyze the prevalence of Clostridioides difficile and its toxins (A/B) in the feces of domestic animals at a University Veterinary Hospital in Teresina - PI. The detection of C. difficile and its toxins was performed by an immunogenic enzyme, called C. Diff Quik Chek Complete® (TECHLAB), capable of detecting antigen glutamate dehydrogenase (GDH) and A/B toxins produced by this bacillus, performed in fecal samples of dogs (C. lupus) and cats (Felis catus) collected between August 2019 and September 2020.:54 stools were analyzed, of which 16 were positive for C. difficile (29.63%). 68.75% (11/16) belonged to canines, while 3.25% (5/16) to felines. Diarrheal and non-diarrheal diseases are used for the study and a higher prevalence of bacillus can be identified in diarrheal diseases (33%). None of the samples present pathogen toxins. The results of this study show that C. difficile is present in the state of Piauí. It can be identified in all species and in diarrheal or non-diarrheic samples, demonstrating that this infection can be symptomatic and asymptomatic, giving the possibility of cross-infection between the animal and its owner.

Bezlotoxumab:A Novel Agent for the Treatment of Clostridium Difficile Infection / 中国药师

Bezlotoxumab is a human monoclonal antibody that can bind to C. difficile toxin B and neutralize its effects. In October 2016, bezlotoxumab was approved by the food and drug administration(FDA) to reduce the recurrence of Clostridium difficile infection (CDI) in the patients aged equal or above 18 years who are receiving antibacterial therapy. This paper introduced the pharmacology, pharmacokinetics,clinical studies,adverse reactions,interactions and medication attentions of bezlotoxumab.

Performance of chromID Clostridium difficile Agar Compared with BBL C. difficile Selective Agar for Detection of C. difficile in Stool Specimens

We evaluated the performance of a new chromogenic medium for detection of Clostridium difficile, chromID C. difficile agar (CDIF; bioMerieux, France), by comparison with BBL C. difficile Selective Agar (CDSA; Becton Dickinson and Company, USA). After heat pre-treatment (80degrees C, 5 min), 185 diarrheal stool samples were inoculated onto the two media types and incubated anaerobically for 24 hr and 48 hr for CDIF and for 48 hr and 72 hr for CDSA. All typical colonies on each medium were examined by Gram staining, and the gram-positive rods confirmed to contain the tpi gene by PCR were identified as C. difficile. C. difficile was recovered from 36 samples by using a combination of the two media. The sensitivity with CDIF 48 hr was highest (100%) and was significantly higher than that with CDIF 24 hr (58.3%; P<0.001), because samples with a low burden of C. difficile tended to require prolonged incubation up to 48 hr (P<0.001). The specificity of CDIF 24 hr and CDIF 48 hr (99.3% and 90.6%, respectively) was significantly higher than that of CDSA 48 hr and CDSA 72 hr (72.5% and 67.1%, respectively; P<0.001). CDIF was effective for detecting C. difficile in heat-pretreated stool specimens, thus reducing unnecessary testing for toxin production in non-C. difficile isolates and turnaround time.

Lactobacillus rhamnosus GG Protects Cells from Clostridium difficile Toxins.

Aims: To determine the anti-cytotoxic effects of Lactobacillus rhamnosus GG (LGG) against extracellular and intracellular Clostridium difficile toxins. Study Design: Co-culture system. Place and Duration of Study: Division of Infectious Diseases, Department of Medicine, School of Medicine and Public Health and Department of Pathobiological Sciences, School of Veterinary Medicine, Madison, Wisconsin, between April 2010 and August 2011. Methodology: In this study, we investigated the effects of a probiotic LGG (Culturelle®) against a toxigenic C. difficile strain (ATCC 9689) and a non-toxigenic C. difficile strain (ATCC 700057) in a co-culture system. Co-cultures were prepared with 3 ml of 1:10, 1:100 or 1:1000 dilution of an overnight culture of LGG and 2 ml of 1:100 dilution of either the toxigenic or the non-toxigenic strain. Cytotoxic effects of cell-free culture supernatants (CFS) and cell lysates of the toxigenic strain on Vero cells were evaluated after coculturing. The relative abundance of toxin A (TcdA) and Toxin B (TcdB) genes in 72 h cocultures were determined using real time PCR. Results: In co-cultures with 1:10 or 1:100 dilution of LGG, counts of the toxigenic C. difficile strain were about one log unit lower than control pure cultures after incubation for 48 h. In all co-cultures, counts of the non-toxigenic strain were two log units lower than those of controls. Accordingly, LGG resulted in a significant decrease (p < 0.05) in the relative abundance of TcdA and TcdB in target DNA prepared from co-cultures containing the 1:10 or the 1:100 dilution of the probiotic. Co-culturing the toxigenic strain with the probiotic (1:10 and 1:100) decreased (P < 0.05) the cytotoxic effect of both extracellular and intracellular clostridial toxins resulting in up to 30% increase in cell viability. Conclusion: LGG inhibits the growth of C. difficile in a dose-dependent manner and protects cells from C. difficile induced cytotoxicity.

Two Cases of Refractory Pseudomembranous Colitis that Healed Following Fecal Microbiota Transplantation / 대한내과학회지

The incidence, recurrence, and mortality of Clostridium difficile infection are increasing and the standard therapy is oral metronidazole or vancomycin. Since treatment failure with standard therapy is increasing, an alternative therapy is needed. Fecal microbiota transplantation is one effective method in patients with refractory or recurrent C. difficile infection, including pseudomembranous colitis. Here, we report two cases of refractory pseudomembranous colitis treated with fecal microbiota transplantation.

Effect of biotherapeutics on antitoxin IgG in experimentally induced Clostridium difficile infection.

Purpose: Recurrent diarrhoea after successful treatment of primary Clostridium difficile associated disease (CDAD) occurs due to bowel flora alterations and failure to mount an effective antibody response. Apart from antibiotics, risk factors include immunosuppressive and acid-suppressive drug administration. Biotherapeutics such as probiotic and epidermal growth factor (EGF) may offer potential effective therapy for CDAD. Materials and Methods: The effect of biotherapeutics in mounting an antibody response against C. difficile toxins was studied in BALB/c mice challenged with C. difficile after pre-treatment with ampicillin, lansoprazole or cyclosporin. Sera from sacrificed animals were estimated for antitoxin IgG by enzyme linked immunosorbent assay. Results: Antitoxin IgG was significantly higher (P<0.05) in C. difficile challenged groups compared to unchallenged controls, but insignificant (P>0.05) in animals in which C. difficile was given after pre-treatment with cyclosporin compared to those without any pre-treatment, or pre-treatment with antibiotic or lansoprazole. In inter-subgroup comparisons also significant anomaly in production of antitoxin IgG was found. The antitoxin IgG levels were raised in animals administered C. difficile after pre-treatment with ampicillin, but lower in animals administered cyclosporin. High levels of antitoxin IgG were also found in the serum samples of animals receiving lansoprazole and C. difficile. Conclusions: Probiotics showed their beneficial effect by boosting the immune response as seen by production of antitoxin IgG. Oral administration of EGF did not affect the immune response to C. difficile toxins as significant increase was not observed in the serum antitoxin IgG levels in any of the groups investigated.

Preliminary investigation of environmental prevalence of Clostridium difficile affecting inpatients in a north Indian hospital.

A preliminary study was conducted to see the prevalence of Clostridium difficile in patients and their environment in a tertiary care hospital. Seventy-nine fecal specimens from hospitalized patients, 176 swab samples from beds and 48 from hands of hospital personnel were investigated. Sixty-three patients received antibiotics and 14 proton pump inhibitors. Abdominal pain was observed in 16 patients with fever in 15 of them. C. difficile culture was positive in 12.6% patients at initial sampling but none were toxin-positive. Eight patients developed diarrhea and five were both culture and toxin-positive. Fifty-one percent of bed swab samples and 62.5% of hand swab samples were culture positive. Similarly 8.5% of bed swab samples and 4.2% of hand swab samples were positive for toxins A and B. The environmental cross-infection between patients and carriage by hospital personnel are plausible sources of C. difficile infection and spread in our hospital.

Evaluation of a ChromID C. difficile Agar for the Isolation of Clostridium difficile / 대한임상미생물학회지

BACKGROUND: Clostridium difficile is the main etiologic agent of antibiotic-associated diarrhea and the most common cause of hospital-acquired diarrhea. Recently, the incidence of C. difficile infections (CDI) has increased and new highly virulent C. difficile strains have emerged. Therefore, accurate and rapid diagnosis is needed. We compared the results of using chromID C. difficile (chromID CD, bioMerieux, France) with the conventional C. difficile Selective Agar (CDSA; BD, USA) for the isolation of C. difficile. METHODS: A total of 738 stool specimens of suspected CDI patients at the Severance Hospital from July to August 2011 were inoculated onto CDSA. Among them, 104 stool specimens revealed colonies on CDSA that were then re-inoculated onto chromID CD. The stool samples were stored at -20degrees C until the time of the re-inoculation. Cultured agars were interpreted after 24 hrs and 48 hrs, respectively. Species identification was performed on the basis of colony characteristics on agar plates as well as the ATB 32A system (API System SA, France). RESULTS: The recovery rates of CDSA and chromID CD were 30.1% and 77.5% after 24 hrs, and 77.5% and 98.6% after 48 hrs, respectively. All of the C. difficile isolates were recovered as typical gray/black colonies on chromID CD. CONCLUSION: The performance of chromID CD for the isolation of C. difficile was better than that of conventional CDSA. The chromID CD could provide easy and sensitive detection of C. difficile even after 24hrs of incubation.

Influence of a Probiotic Milk Drink, Containing Lactobacillus Paracasei Lpc-37, on Immune Function and Gut Microbiota in Elderly Subjects.

Immunosenescence and alterations in the intestinal microbiota are associated with aging. Immune functions, as well as the intestinal microbiota, can potentially be modified and improved by probiotics, thus being particularly beneficial to elderly consumers. The ability of Lactobacillus paracasei Lpc-37 to modulate immune markers including phagocytic activity, natural killer (NK) cell activity and cytokine profiles, as well as composition and activity of the intestinal microbiota, in healthy elderly subjects was investigated in a randomised, double-blind, placebo-controlled study. Only very limited effects in the measured blood or faecal immune markers or intestinal microbiota could be detected between the fermented milk drinks with or without probiotics. Thus, no significant immunological or microbial effects of the probiotic fermented milk could be detected in this study population.

The role of flexible sigmoidoscopy in the diagnosis of Clostridium difficile-associated diarrhea / 대한내과학회지

BACKGROUND/AIMS: Clostridium difficile is an important cause of diarrhea in hospitalized patients. C. difficile-associated diarrhea (CDAD) is usually diagnosed following a stool test for C. difficile cytotoxin or stool culture for the presence of toxigenic C. difficile. However, the reported sensitivities of these tests are variable. Sigmoidoscopy may be an effective diagnostic method in patients with a false-negative stool test for cytotoxin. This study examined the role of flexible sigmoidoscopy in the diagnosis of CDAD. METHODS: Among the patients who had diarrhea and were examined with sigmoidoscopy in Eulji University Hospital between January 2005 and July 2008, 102 patients suspected of having antibiotic-associated diarrhea (AAD) based on their clinical symptoms were enrolled. Of the 102 patients, 74 were diagnosed with CDAD based on C. difficile cytotoxin or sigmoidoscopic findings of pseudomembranous colitis. The medical records of these 74 patients were reviewed retrospectively. RESULTS: Of the 74 patients, sigmoidoscopic findings revealed a pseudomembrane in 63 patients (85.1%) and colitis in nine (12.2%), while two patients (2.7%) appeared normal. Of the 63 patients with pseudomembranous colitis at sigmoidoscopy, the stool C. difficile cytotoxin assay was negative in 27 (42.9%). CONCLUSIONS: Flexible sigmoidoscopy was highly sensitive in pseudomembranous colitis and is useful in diagnosing patients with a delayed or negative stool test for C. difficile cytotoxin. Therefore, we recommend flexible sigmoidoscopy in patients suspected of having C. difficile-associated diarrhea for the diagnosis of CDAD.

Established and potential risk factors for clostridum difficile infection.

Clostridium difficile is the aetiological agent for almost all cases of pseudo membranous colitis and 15-25% of antibiotic associated diarrhoea. In recent years, C. difficile associated disease (CDAD) has been increasing in frequency and severity due to the emergence of virulent strains. Severe cases of toxic mega colon may be associated with mortality rates of 24-38%. The prevalence of CDAD is global and the incidence varies considerably from place to place. In the initial stages of its discovery, C. difficile infection was regarded mainly as an outcome of antibiotic intake and not as a life threatening disease. Intervention by man has produced conditions making C. difficile a significant cause of morbidity and mortality. The recent outbreak of CDAD in Quebec has sent the alarm bells ringing. Apart from a threefold increase in the incidence of CDAD, clinicians have also reported a higher number of cases involving toxic mega colon, colectomy or death. Among all the risk factors, inclusive of the host and the environmental factors, antibiotics are the most important ones. Surgical patients comprise 55-75% of all patients with CDAD due to the fact that perioperative prophylaxis requires the use of antibiotics. However, other drugs such as immunosuppressants and proton pump inhibitors are also important risk factors. Thus CDAD is a growing nosocomial and public health challenge. Additionally, the recognition of community acquired CDAD signals the presence of several risk factors. In this review, the established and potential risk factors of CDAD, along with the epidemiology, diagnostic modalities, management and preventive measures of the disease have been elaborated.

Antibiotic associated diarrhea in children.

Context: Keeping in view the recent flooding of the Indian market with antibiotic and probiotic combinations, we decided to look at the prevalence of antibiotic associated diarrhea (AAD) and Clostridium difficile infection (CDI) in children and reviewed evidence available for use of probiotics in the prevention of AAD. Evidence acquisition: We did a PubMed, Medline and Cochrane libary search for literature available in last 25 years. Results: Prevalence of antibiotic associated diarrhea (AAD) is around 11%. Children younger than 2 years and type of antibiotics are the two risk factors identified for AAD. For the pediatric population, CDI reportedly decreased in a tertiary care hospital in India, though number of suspected samples tested increased. The incidence of community acquired CDI is increasing in the pediatric population also. Detection of toxin A and B by enzyme linked immunosorbent assay (ELISA) and detection of toxin B by tissue culture form the mainstay in the diagnosis of C. difficile. Most of the AAD would respond to only discontinuation or change of the antibiotic. Oral metronidazole or oral vancomycin are drugs of choice for CDI. Probiotics reduce the risk of AAD in children and for every 7-10 patients one less would develop AAD. Conclusion: Prevalence of AAD is low and majority will respond to discontinuation of antibiotic. CDI is uncommon in children. Probiotics will prevent AAD in only 1 in 7 children on antibiotics. We need cost effectiveness studies to decide the issue of needing a probiotic antibiotic combination to prevent AAD.

Management of Antibiotics-Associated Diarrhea / 대한소화기학회지

Antibiotics-associated diarrhea (AAD) is defined as unexplained diarrhea that occurs with the administration of antibiotics. Approximately 20% AAD cases are due to Clostridium difficile. Over the last decade, the incidence of Clostridium difficile-associated disease (CDAD) has progressively increased, and now a significant clinical problem. Recent change in the epidemiology of CDAD and the emergence of an epidemic hypervilruent strain suggest the need for greater attention for infection control, early diagnosis, and more effective treatment modality. However, since most cases of CDAD are both iatrogenic and nosocomial, careful selection of antibiotics, combined with proper hand hygiene and precaution by medical staffs are required.

Clinical Characteristics and Changing Epidemiology of Clostridium difficile-Associated Disease (CDAD) / 대한소화기학회지

BACKGROUND/AIMS: The spectrum of Clostridium difficile-associated disease (CDAD) ranges from mild diarrhea to life-threatening colitis. Recent studies reported an increase in incidence and severity of CDAD and the presence of severe community-acquired CDAD (CA-CDAD). The aims of this study were to investigate the incidence of CA-CDAD and non-antibiotics-associated CDAD, and to compare the clinical characteristics between hospital-acquired (HA) and CA-CDAD. METHODS: The medical records of 86 patients who were diagnosed as CDAD in Hanyang University Guri Hospital between January 2005 and October 2007 were retrospectively reviewed. RESULTS: Of the 86 patients (mean age 64 years), 53 patients were women. The most frequently prescribed antibiotics were cephalosporins (67.4%), followed by aminoglycosides (38.4%) and quinolones (14%). Of the 86 patients, the average duration of treatment and recovery time of symptoms were 11.5 days and 4.6 days, respectively. Seven percent of patients experienced relapse treatment. The overall incidence rate of CA-CDAD and non-antibiotics-associated CDAD were 10.5% and 22.1%, respectively. CA-CDAD group had lower rate of antimicrobial exposure whilst showing higher rate of complications compared to HA-CDAD group. Three patients in the CA-CDAD progressed towards a severe complicated clinical course, including septic shock. CONCLUSIONS: The incidence rate of CA-CDAD and non-antibiotics-associated CDAD were 10.5% and 22.1%, respectively. CA-CDAD tends to have a higher complication rate compared to HA-CDAD. Community clinicians needs to maintain a high level of suspicion for CDAD, whilst coping with the ever evolving epidemiologic change.

Nuclear Factor-kappa B Activation and Chemokine Genes Expression in HT-29 Intestinal Epithelial Cells in Response to Clostridium difficile Toxin A Stimulation

Intestinal epithelial cells are known to up-regulate the expression of several chemokines in response to bacterial toxins. Since there has been little understanding on the cellular mechanisms of C. difficile toxin A-induced mucosal inflammation, we investigated whether nuclear factor-kappa B (NF-kappaB) could regulate chemokine gene expression in HT-29 intestinal epithelial cells stimulated with C. difficile toxin A. C. difficile toxin A rapidly increased signals of NF-kappaB composed with p65 and p50 subunits in HT-29 cells, whereas it decreased the signals of IkappaBalpha. Blocking the NF-kB activation by transfection with dominant negative I kappa B alpha-containing retrovirus attenuated the upregulated expression of IL-8, GRO-alpha, and MCP-1 induced by C. difficile toxin A. These results suggest that NF-kappaB is a major regulator of chemokine gene expression in C. difficile toxin A-stimulated intestinal epithelial cells.

Molecular Analysis of Clostridium difficile Isolates by Arbitrarily Primed-Polymerase Chain Reaction and Polymerase Chain Reaction-Ribotyping / 감염

BACKGROUND: Clostridium difficile is known as the major cause of nosocomially acquired diarrhea. Various phenotypic and genotypic methods have been used to subtype C. difficile strains. The purpose of the present study is to evaluate several typing methods which can be used as tools for subtyping C. difficile isolates for epidemiological studies. METHODS: In two Korean tertiary care hospitals, a total of 81 C. difficile isolates were collected from symptomatic, hospitalized patients in 1998. All isolates were examined for the release of toxin A and toxin B by PCR assay and cell culture assay. Also arbitrarily primed-PCR and PCR-ribotyping profiles were determined for the typing of C. difficile strains on a genetic level. RESULTS: The toxin B gene was detected in 65.4% (54/81) of isolates by both PCR assay and cell cultureassay. Nine types were identified with T-7 primer, and 13 types were identified with PG-05 primer in AP- PCR. Sixteen types were identified in PCR-ribotyping. When two typing methods were compared, reproducibility by PCR-ribotyping was 100%, while it was only 83% and 33% AP-PCR with primer T-7, and PG-05, respectively. The discrimination index was 0.88 for PCR-ribotyping, 0.82 for AP-PCR with primer T-7 and 0.81 with primer PG-05. CONCLUSION: These data suggest that PCR-ribotyping provides a reproducible, discriminatory, and simple alternative to conventional molecular approaches for typing strains of C. difficile.

Evaluation of Quantitative culture of Clostridium difficile From Fecal Specimens for the Diagnosis of C. difficile-associated Disease / 대한임상미생물학회지

BACKGROUND: C. difficile-associated diarrhea (CDAD), the most frequently identified cause of nosocomial diarrhea, results from the overgrowth of cytotoxin (toxin B)-producing strains. The aim of this study was to evaluate the quantitative culture of Clostridium difficile to improve the laboratory diagnosis of CDAD. METHODS: The quantitative culture and cytotoxin gene results were evaluated based on the findings of colonoscopy and/or histology of the biopsy specimens. RESULTS: Among the 402 specimens with cytotoxin-positive isolates, 301 (74.9%) contained > or =106 CFU/mL of C. difficile. Nine (60%) of the 15 pseudomembranous colitis patients yielded > or =106 CFU/mL of toxigenic isolate. The proportion of cytotoxin gene-positive isolates was higher in the specimens with > or =106 CFU/mL of C. difficile than in those with 102-<103 CFU/mL (86.5% vs. 66.7%). CONCLUSIONS: Quantitative culture may aid in the interpretation of toxigenic C. difficile culture results, and reduce false positivity, thus avoiding unnecessary treatment.