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1 Edit Urbán Department of Clinical Microbiology Faculty of Medicine University of Szeged Hungary

2 History Microbiology and ecology Epidemiology Transmisson Pathogenesis Clinical manifestations Diagnosis Therapy

3 1893 Finney description of PMC 1935 Hall and O Toole first description of C. difficile 1974 Tedesco PMC linked to antibiotic use 1974 Hafiz presence of C. difficile in environmental sites Larson cytopathic effect of stools of PMC patients Rifkin et al. toxin in the stool of PMC patients that was neutralised by C. sordellii antitoxin 1978 Bartlett toxigenic clostridia were the cause of PMC 1978 Larson et al. isolation of C. difficile from stool of PMC patients 1981 Vancomycin approved by FDA for CD 1982 Metronidazole for CDI 1984 ELISA-s for toxin detection Outbreak in US and Europe *John Finney and Sir William Osler: Gastro-enterostomy for cicatrizing ulcer of the pylorus. Bull Johns Hopkins Hosp. 4;

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5 Clostridium difficile május 15. Szeged

6 Strict anaerobe Gram positive rod Subterminal elongated spores Irregular, flat, rough colonies Fluoresce chartreuse Bacillus difficilis Selective agars (CCFA)

7 Environmental source Possible community sources for CDI Soil, water, waste water, manure, pets, meats, vegetables

8 20-40% carriers Animal reservoirs május 15. Szeged

9 Vegetative vs. spore forms

10 Spores: Extremely hardy, survive for long amounts of time in environments Resistant to drying and heating, and to many forms of antiseptic cleaners Can also survive in spore form for as long as five months The ability of C. diff. to survive in this resistant form poses quite a challenge for hospitals Vegetative form: Change into its vegetative, disease causing state when it reaches the intestine of humans. Human intestine also contains glycine and cholate derivatives, which are the two substances that C. diff requires to germinate In healthy intestines with a normal population of microbes, cholate derivatives are further processed by the other bacteria present, thus preventing germination of the foreign C. diff After broad-spectrum antibiotic treatment, much of the microflora of the intestine is disrupted or killed; this prevents the further processing of cholate, and allows C. diff. to germinate Gives C. diff. more space in the intestine, as much of the other microbes that would ordinarily be in the intestine have been destroyed by the antibiotic treatment David A. Burns, John T. Heap, Nigel P. Minton. Clostridium difficile spore germination: an update. Research in Microbiology. Volume 161, Issue

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12 GLC Acetic Acid Butyric Acid Isobutyric Acid Isovaleric Acid Isocaproic Acid Presumptive traditional identification: Biochemical tests Smell UV fluorescence RapID ANA 4 hour enzyme detection system 10 reaction cavities 18 tests ATB ID 32A REMEL Rosco disks Latex Agglutination MALDI-TOF

13 Normal fecal flora of newborns: 60-70%- asymptomatic Most important nosocomial enteric pathogen Asymptomatic carriers: (Healthy adults: transient) Eu: % Japan: 15 % Hospitals, nursing homes: 10-20% of elderly individuals colonized 20-40% of hospitalized patients colonized Nurses? GP? MD? Nosocomial clusters Isolated from toilets, bedpans, floors Spores isolated for up to 6 months Hookman P, Barkin, JS. World J Gastroenterol. 2009;15: Makris AT, Gelone S. J Am Med Dir Assoc. 2007;8: Cohen SH, et al. Infection Control and Hospital Epidemiology. 2010;31(5): Goodhand JR, et al. Ailment Pharmacol Ther. 2011;33: Aseeri M, et al. Am J Gastroenterol. 2008;103:

14 In 2003, in Southern Quebec and the Montreal district of Can. an epidemic of C. difficile disease (CDD) was recorded By January 2005, 30 hospitals in Quebec were reporting rates of CDI at five times their normal level This strain has now been identified in several US states PCR ribotype 027 and has been assigned as PFGE Type NAP1describe the clonal international strain (NAP1/027) Produced 8 to 16 fold greater amounts of toxins A and B in vitro in compared to other strains This would tie in with clinical information of more severe disease and increased death rates seen in outbreaks with this strain. Hyper-toxin production was believed to be due to an 18bp deletion in toxin the TcdC gene that regulates toxin production Binary toxin High level quinolon resistant Exposure of epidemic strains to sub-inhibitory concentration of non-chlorine-based cleaning agents higher sporulation capacity in vitro The Lancet 24thth Sept 2005 Warny, Pepin, Fang, Killgore, Thompson, Brazier, Frost and McDonald: Toxin production by an emerging strain of C. difficile associted with outbreaks of severe disease in North America and Europe

15 In late June 2005, Dr.Ed Kuijper from Leiden University Medical College gave information about of a serious outbreak of C. difficile infections in a Dutch hospital Isolates found Type 027 was the cause Shortly afterwards, a second Dutch hospital was involved due to a known 027 patient being transferred A few weeks later another hospital in Amsterdam was also identified In October, a Belgian hospital became affected In the UK, major outbreaks emerged in 2006 where the PCR ribotype 027 was identified The proportion of 027 strains isolated in UK hospitals rose sharply from 25.9 % to 41.3 % between 2005 and 2008 Kuijper, E. J., Coignard, B., Brazier, J. Suetens, C., Drudy, D., Wiuff, C.,Pituch, H., Reichert, P. & Schneider, F. (2007): Update of Clostridium difficile-associated disease due to PCR ribotype 027 in Europe.Euro Surveill 12, E1 E2. Brazier, J. S., et al. HPA Regional Microbiology Network (2008).Distribution and antimicrobial susceptibility patterns of Clostridium difficile PCR ribotypes in English hospitals, Euro Surveill 13,

16 Significant increase in worldwide incidence of CDI Changes in the profile of populations for acquiring CDI Asensio et al. Eurosurv. (2008)

17 Lucado et al.

18 CDI incidence in USA the number of CDI cases appears to be increasing. According to the Nationwide Inpatient Sample of the Healthcare Cost and Utilization Project, the total number of patients assigned the ICD-9 code for CDI (008.45) in acute care facilities has increased from 138,954 in 2000 to nearly 350,000 in , ,950 Healthcare Cost and Utilization Project (HCUP).

19 20,4/1 000 discharges 15,2/ ,29/ ,97/1 000 Healthcare Cost and Utilization Project (HCUP).

20 Annual incidence (per population) of Clostridium difficile-associated diarrhea (CDAD) in Sherbrooke, Que., Pepin, J. et al. CMAJ 2004;171:

21 Increased incidence largely attributed to the emergence of PCR ribotype 027 (NAP1/BI) Clements et al. Lancet Infect Dis (2010)

22 After large outbreaks caused by PCR027, PCR ribotypes other than 027 were most prevalent in Europe UK PCR ribotype 106 The Netherlands PCR ribotype 078

23 EPINFO 18. évf. 4. különszám augusztus 09.

24 Statement obligatory 2011-law

25 Severity of CDI appears to be increasing Severe CDI reported in postpartum woman Associated with cesarean delivery, antibiotic use, and choriamnionitis Increasing evidence of CDI in paediatric population More severe presentations, emergence of hypervirulent strains Garey et al. Am J Obstet Gynecol (2008) CDC MMWR Weekly (2005)

26 Higher rate of community-onset CDI CDI with onset in the community or in a health care facility within 48 h after admission and the patient has not been discharged from a health care facility in the previous 12 weeks 2005 CDC received 23 report of CDI in outpatients with no serious underlying disease No hospital exposure in the previous 3 months 26% required hospitalisation, 48% <age 18 years, 35% no previous antibiotics Diarrhoeal disease in close contact Hospital admission Discharge 48 h <4 weeks 4-12 weeks Healthcare onset Community onset Time * Health-care associated Unknown Community associated CDC MMWR Weekly (2005)

27 3 important factors: Alteration of normal gut flora Exposure of toxin producing C. difficile strain digest, lower GI tract At least one of other factors Advanced age 65 yrs Serious underlying diseases Hospitalisation Immunsuppression IBD Interventions of GI tract (NG tubes, sondas, surgery, ) Use of histamine-2 receptor blockers or proton-pump inhibitors

28 Poutanen SM, Simor AE. Can Med Assoc J. 2004;171(1):51-58.

29 Toxins: Glucosyltransferases Bind to specific receptors on the colonic epithelium Rounding of cells Membrane blebbing Apoptosis Transported into the cytoplasm Both toxins inactivate Rho proteins GTP-binding proteins important in actin polymerization, cytoskeletal architecture and cell movement Disruption of the actin cytoskeleton Disruption of epithelial cell tightjunction proteins A toxin (308 kda) responsible for the enteric symptoms enterotoxin (cytotoxin) Chemoattractant of neutrophils Macrophage activator B toxin (269 kda) cytotoxin Actin-cytoskeleton disruption Potent necrotizing enterotoxin Sinergistic with toxin A Binary toxin: 2 component ADP-ribosyltransferase activity >80% homology with C. perfringens iota toxin In 6.4% of isolates from the UK Due to genetic exchanges among several clostridial species Appears to enhance pathogenicity of NAP 1/O27

30 Role of binary toxin unclear Binary toxin possesses cytotoxic activity in vitro (Sundryial et al. 2010) Increased adherence of bacteria to target cells by the formation of microtuble protrusions Other non-toxigenic pathogenecity factors: Adherence and intestinal colonisation (66-kDa cell-wall protein CWp66, GroEL heat-shock protein, 68 kda fibronectinbinding protein, flagella components FliC and FliD) Cysteine protease Cwp84 Proteolytic activity toward several proteins of EC matrix Role in the maturation of S-layer S-layer Binding in epithelial and connective tissue (largely restricted to apexes of villi) Chapeton Monets et al. J Bacteriol (2011)

31 Toxin A + Toxin B +, binary toxin + Toxin A + Toxin B +, binary toxin Toxin A - Toxin B +, binary toxin + Toxin A - Toxin B -, binary toxin + Toxin A - Toxin B -, binary toxin Toxin A+ Toxin B-, binary toxin - Toxinotypes 0, I-XXVII.

32 Clinical Microbiology Reviews 18:

33 Pseudomembranous colitis (PMC) % Antibiotic associated colitis (AAC) 60-75% Antibiotic associated diarrhoea (AAD) 11-33% In hospital settings: asymptomatic carriers 2-5x more frequent, than CDI Gerding DN, et al. Infect Control Hosp Epidemiol. 1995;16: CDC. Fact Sheet, August 2004 (updated 7/22/05). McDonald LC, et al. Emerg Infect Dis. 2006;12:

34 Disease shown caused by a filterable agent That agent could be neutralized by antibodies against clostridial toxins Toxin identified as produced only by Clostridium difficile Hamster model with characteristic dark red inflammation of small intestine

35 Diarrhea within a few days of antibiotic therapy Faeces have a distinctive foul odour Abdominal pain +/- pyrexia Blood if pseudomembranous colitis Electrolyte disturbances Hypoalbuminaemia Paralytic ileus Toxic Megacolon/perforation/shock Death Increased WBC

36 Diagnosis must be confirmed by laboratory tests Not all AAD is CDI Sample selection CDI testing performed on unformed stool In patients with infective diarrhoea, negative tests for enteropathogens, irrespective of age, prior antibiotic use, comorbidity and onset of diarrhoea In patients with diarrhoea who have been hospitalised more than 72 h irrespective of physicians request, or who have been admitted to health-care facility within a period of 3 months

37 Test Advantages Diadvantages Toxin testing Enzyme immunoassay Tissue culture cytotoxicity Organism identification Detection of glutamate dehydrogenase PCR Rapid, simple, inexpensive More sensitive than enzyme immunoassay Rapid, sensitive, may prove useful as a triage or screening tool Rapid, sensitive, detects presence of toxin gene Least sensitive method, some detect only toxin A (some strains only produce toxin B) Labor intensive; requires hours for a final result, special equipment; not as sensitive as generally thought Not specific, toxin testing required to verify diagnosis; may not be 100% sensitive Cost, special equipment, may be too sensitive Stool culture Most sensitive test available when performed appropriately May be associated with false-positive results if isolate is not tested for toxin; labor-intensive; requires hours for results

38 On the basis of clinical hallmarks and microbiological tests Detection of toxins A and B cornerstone of laboratory diagnosis Cell cytotoxicity assay (CCNA) from faecal supernatant or culture -reference standard Time consuming, labour intensive EIA for toxin detection generally low sensitivity Two-step (three-step) protocol recommended to detect glutamate dehydrogenase and free toxins in faecal specimen Initial screen: EIA for glutamate dehydrogenase Confirmatory: cell cytotoxicity assay (or culture) or polymerase chain reaction (PCR) Crobach et al., CMI (2009)

39 Many molecular methods are in-house assays Need DNA purification step to eliminate PCR inhibitors in faecal samples 16S rrna and GDH genes Differentiation between toxigenic and nontoxigenic strains is not possible Real-time PCR Primer pairs and molecular bacon probes were designed to the conserved region of the tcda, and tcdb + tcdc, and cdtb genes Rapid method (about min) The detection limit is about 10 genome copies per PCR in the case of pure culture The detection limit is about 5x10 4 CFU/g of faeces Does not need post-pcr analysis Sensitivity 97% (from faecal specimen), specificity 100% (from faecal specimen)

40 Assay Target Extractio Method BD GeneOhm Cdiff tcdb Manual Xpert C. difficile tcdb, cdta, tcdc deletion PCR-molecular bacon Time (DNS extraction) 2-3 h Automata PCR-Taqman 1 h illumigene C. difficile tcda Manual LAMP 1 h Assay Sensitivity Specificity BD GeneOhm Cdiff Xpert C. difficile illumigene C. difficile 95.5% 97.9% 97.8% 97.9% 86.7% 100% Equipment Smart Cycler (Cepheid) GeneXpert (Cepheid) illumipro-10 (Meridian) Viala et al., JMM (2012) 90: INC_8-K/c98174exv99w1.htm#b

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42 Screen GDH or PCR (tcdb) Confirmation Direct/indirect CCNA/toxin EIA/tcdB PCR From the same sample Only from the positive screen test PCR most sensitive (~98%) CCNA (~66%) Toxin A/B EIA (~42%) EIA low sensitivity CCNA long time Screening test negatíve predictive value very high eredmény interpretálható Culbreath et al., JCM (2012) 50:

43 Screen GDH PCR (tcdb) from all positive samples PCR detection limit 1x10 5 /gramm faeces Reference: toxigenic culture senzitivity 94%, specificity 99%

44 Screen GDH Toxin A/B ICD CCNA/NAAT/toxigenic culture, confirmation 90%- of samples same results with GDH/CCNA algorithm In other 10%-confirmatory tests Goldenberg et al. (2010): This algorithm achieves a reasonable balance between accuracy, rapidity and cost, and will be attractive to many clinical laboratories. Goldenber et al., J Hosp Infect (2010) 74: Larson et al., J Clin Microbiol (2010) 48:

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46 Discontinue offending antimicrobial agent (if possible) Send stool specimen for C. difficile testing Initiate CDI therapy either empirically or following confirmation of diagnosis (depending on clinical urgency) Pharmacotherapy Vancomycin (only FDA-approved treatment for CDI) Metronidazole Supportive treatment Monitor for symptom resolution Cohen et al. Infect Control Hosp Epidemiol (2010)

47 Clinical scenario Supportive clinical data Recommended treatment Mild to moderate Leukocytosis (WBC < 15,000 cells/ul) or SCr level < 1.5 times premorbid level Severe Leukocytosis (WBC 15,000 cells/ul) or SCr level 1.5 times premorbid level Severe, complicated Hypotension or shock, ileus, megacolon Metronidazole 500 mg 3 times per day PO for days Vancomycin 125 mg 4 times per day PO for days Vancomycin 500 mg 4 times per day PO or by nasogastric tube plus metronidazole 500 mg IV q 8 hrs Cohen et al. Infect Control Hosp Epidemiol (2010)

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