Microsoft word - xinix resultat 080513 tn, eng.

Inactivation of spores of Clostridium difficile with chlorine dioxide (SanDes®)
Background
Clostridium difficile (CD) is a spore forming bacteria that normally is present in the intestine of 1-3% of human beings. When treated with antibiotics the presence of CD is 10-20%. CD is a bacteria that can cause diarrhoea1. CD has most likely a low infective dose and is in high degree faeces and orally infectious via direct and indirect contact2,3. When CD bacteria is discovered in faeces approximately 10% consists of spores. The spores may live for months in the environment around a patient, in a contaminated hospital room and may infect a patient newly admitted to hospital with CD, if the patient is treated with antibiotics2. Infection may also take place from an infected patient to another patient and cause colony build-up in fellow-patients within the same care department, which makes it to a nosocomial intestine In Sweden is spread of CD a fast growing problem within the health care. The ways for contamination are difficult to substantiate but most likely the contamination takes place via indirect contagion, for instance via the hands of personal, gloves or other objects4. In 1979 in Sweden 86 cases were diagnosed with a steady increase noticed that grew to 533 cases in 19842. The diagnostics was thereafter centralized causing lack of continuous national statistics. The problem grew and January 1, 1995, the Swedish Institute for Infectious Disease Control (SMI) initiated a study to determine the number of incidents. The study showed that more than 6000 patients in Sweden were CD positive. In 1955 there were in Sweden as an average 50 cases per 100 000 inhabitants and year. In Örebro the number of incidents was higher than the national average, showing 60 cases per 100 000 inhabitants and year. Half of the infected cases in Örebro were initiated in hospital5. In 2004 a CD study was performed in the Örebro county. At that time the average number of cases was 5,3 for 1000 persons hospitalized. The yearly number of incidents in the county were 97 per 100 000 inhabitants, an increase since 1995. Of the 372 patients 208 patients (78%) were hospital related. Of these 36 patients (17%) were suspected to have been During the fall of 2005 an increase of CD cases was reported from Canada. The number of cases per 100 000 inhabitants had increased from 102 cases during 1991-1992 to 210 cases 2002 and 866 cases in 2003. A new strain of CD, not reported earlier in connection to hospital SanDes is chlorine dioxide and has unlike chlorine no recognized undesired side-effects. Chlorine dioxide is known as a very effective means for inactivation of bacteria. It is used for disinfection of drinking water. Chlorine dioxide has a documented effect against Clostridium perfringens spores8. and for the moment there is no functioning means against Clostridium difficile spores. For this reason we are interested in investigating if SanDes has effect for inactivation of Clostridium difficile spores. To find out whether SanDes can inactivate Clostridium difficile spores. The test was performed January 14-23, 2008, at the Microbiological unit in Örebro by the nurse Maria Sjöberg and biomedical analyzer Josefin Andersson. Physician Torbjörn Norén had the overall responsibility for the project Nutritious clear soup (PY-substance preproduced in anaerobe environment for 24 hours) was added to the test bacteria Clostridium difficile 1067. This mixture was left in anaerobe Two different types of plates for cultivation have been used, FAAAP and TCCFA. These were preproduced (i.e. kept in anaerobe environment) for 3 days before being used. After 6 days in anaerobe environment the amount of spores/vegetative cells was determined with phase contrast microscope in bürker chamber. 20 tubes were filled with 0,1 mL of the Clostridium difficile soup and 4 tubes were filled with staphylococcus (for control). To half of all the tubes PBS 0,9 mL was added and to the remaining tubes EtOH 95% was added. All tubes chock treated with alcohol were left for 1 hour for maximum amount of spores. 6 PBS tubes and 6 EtOH 95% tubes were centrifuged for 40 minutes 3000G4000 rpm. The centrifuged tubes were drained. To every test tube (see table 1) different concentrations of SanDes, except the 0 test, were added for 60 seconds treatment, after which 10 and 300 microliters were cultivated on the two plates. The result shows that at not centrifuged and exposed to 200-800 ppm of SanDes a moderate Log10 reduction between 0,7 and 2.9 (Table 1) was achieved while with 1500 ppm of SanDes a total inactivation of spores was achieved.
Xinix inactivation of Clostridium difficile spores
Inactivation of C. difficile spores in solution
1 min Xinix 200 ppm 40 0 ppm
Table 1.The table shows that exposed to 200-800 ppm of SanDes a moderate Log10 reduction between 0,7 and 2.9 was achieved while with 1500 ppm of SanDes a total inactivation of spores was achieved. Comments
The centrifuged tubes also cause a certain inactivation but with a randomized variation probably because of organic ”packing” in pellets and worse access for SanDes. These results are not presented. The FAAAP plates showed best function in this test. Importance and applicability
The result will have great importance for the elimination of Clostridium difficile spores. The areas for use can be surfaces in hospital rooms and means for hand disinfection. The future
As we have proven inactivation of spores we are proceeding with application for disinfection of surfaces in health care environment and eventually use for hand disinfection. Project description for this tests are in progress. Abbreviations
PY-medium = ”Peptone yeast”-medium with added cystein.
FAAAP = fastidious anaerobe agar (FAA) is a medium for primary isolation of demanding
anaerobic microorganisms. Contains 5% horse blood. TCCFA = taurocholate cycloserine-cefoxitin-fructose-agar. A selective and differentiating
medium that stimulates germination of Clostridium difficile spores. Cycloserine and cefoxitin inhibit growth of most aerobic, anaerobic, and facultative anaerobic gram-positive and gram- negative bacteria. Taurochol acid separates C. difficile from lecitina positive clortides. Clostridium difficile 1067: Epidemic strain B1/NAP1/027 toxinotype III that just now is
spreading in North america and in Europe. PBS: Phosphate buffered saline solution
List of references

1.
Iwarson S, Norrby R. Infektionsmedicin : epidemiologi, klinik, terapi. 3 ed. Sävedalen: Säve; 2004. Clostridium difficile - en "ny" patogen på frammarsch. epidAKTUELLT Gerding DN, Johnson S, Peterson LR, Mulligan ME, Silva J, Jr. Clostridium difficile-associated diarrhea and colitis. Infect Control Hosp Epidemiol 1995;16(8):459-77. Socialstyrelsen. Att förebygga vårdrelaterade infektioner : ett kunskapsunderlag. Stockholm: Socialstyrelsen; 2006. Karlstrom O, Fryklund B, Tullus K, Burman LG. Samhällsförvärvad C difficile-associerad diarré en realitet. Färre recept och mera tvål. Lakartidningen 1997;94(23):2187-92. Norén T, Åkerlund T, Bäck E, Sjöberg L, Persson I, Alriksson I, et al. Molecular epidemiology of hospital-associated and community-acquired Clostridium difficile infection in a Swedish county. J Clin Microbiol 2004;42(8):3635-43. Warny M, Pepin J, Fang A, Killgore G, Thompson A, Brazier J, et al. Toxin production by an emerging strain of Clostridium difficile associated with outbreaks of severe disease in North America and Europe. Lancet 2005;366(9491):1079-84. Foegeding PM, Hemstapat V, Giesbrecht FG. Chlorine Dioxide Inactivation of Bacillus and Clostridium Spores. Journal of Food Science 1986;51(1):197-201.

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