Plasma Science and Technology

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1 ISSN: Plasma Science and Technology Volume 8 Number 4 July 2006 A Publication of Institute of Plasma Physics, Chinese Academy of Sciences and the Chinese Society of Theoretical and Applied Mechanics Distributed by Institute of Physics Publishing Online: INSTITUTE OF PLASMA PHYSICS, CHINESE ACADEMY OF SCIENCES AND THE CHINESE SOCIETY OF THEORETICAL AND APPLIED MECHANICS Institute of Physics PUBLISHING

2 Plasma. Science &- Technology, V01.8. No.4. July 2006 Observation on clinical Sterilization effectiveness of CASP-80 Low temperature Plasma Sterilizer Li SI, Zhang Yangde, LIU Weidong, Xiangya Hospital, Central South University, Changsha ,China Abstract The influence on the sterilization effectiveness of low-temperature plasma sterilizer CASP-80 is explored so as to provide a theoretical for reducing medical costs and achieving sterilization effectiveness, To conduct on-site simulation test, clinical material sterilization test and organic substance influence test respectively by using the representative of Bacillus Stearothermophilus, preparing the bacteria-contaminated carrier through polytetrafluoroethylene (PTFE) simulated hose endoscopes, and using calf serum as the organic substance influence factor. The test result show that CASP-80 low temperature plasma sterilizer and achieve sterilization effectiveness properly through either short-cycle or long-cycle sterilization method according to difference materials, apparatus, and contamination extent. The organic substance could influence the sterilization effectiveness of low temperature plasma sterilizer(h2o2) Keywords: Plasma sterilization, peroxide H2O2 Bacillus steraothermophilus, PTEE hose simulated endoscope influence of organic substance, calf serum. PACS: 52.77Dq.52.77Bn I. Introduce Sterilization is a physical or chemical process that eliminates all life of microorganisms. Conventional sterilization techniques, such as those using autoclaves, ovens, an chemicals like ethylene oxide(eto),rely on irreversible metabolic inactivation or on breakdown of vital structural components of the microorganism [1] Plasma sterilization operates differently because of its specific active agents, which are ultraviolet ( UV) photons and radicals ( atoms or assembly of atoms with unpaired electrons. therefore chemically reactive, e.g., O and OH. respectively) [ 2]. An advantage of the plasma method is the possibility, under appropriate conditions, of achieving such a process at relatively low temperatures ( 50'C ), preserving the integrity of polymer-based instruments. which cannot be subjected to autoclaves and ovens. Furthermore, plasma sterilization is safe. both for the operator and the patient in contrast to [5. 6] ETO Three plasma sterilization tests( on-set simulation, clinical materials and organic substance influence) were carried out based on CASP-80 device which technology is developed by Institute of Plasma Physics in Chinese Academy of Sciences. The test results are presented in this paper and show this technique can be used on endoscopes apparatus sterilization. 2 0n-Site Simulation Test 2.1 Materials CASP-80 low-temperature plasma sterilizer: Provided by Plasma Physics Research Institute of Chinese Academy of Sciences Neutralizer: 0.5% sodium thiosulfate M PBS. bromocresol purple peptone broth, bromocresol purple peptone agar Test bacteria: Bacillus Stearothermophilus bacillus spore (ATCC7953 ) φ1mm (inner diameter)x1000mm (long) PTFE hose, φ0.7mmx40mm steel needle, φ2mmx40mm PTFE hose ( as the casing at junction). 2.2 Methods Preparation of bacteria-contaminated carrier of simulated medical apparatus Prepare test bacteria int CFU/ml bacterial suspension, soak cp0.7mmx40mm steel needle in bacteria suspension for 2h. then take it out to dry in 37'Cincubator for 30min for later use. During the test, put the bacteria contaminated steel needle between the junction of twoφ1mm X1000mm PTFE

3 hose. and seal with casing the interface of two hoses. Pack the sealed hose into special paper bag, seal the bag, put in sterilization cabinet. and make double-cycle sterilization with 50% hydrogen peroxide plasma. Detection method Take out the sterilized carrier in hose. use the positive control of unsterilized carrier. cultivate the sterilized carrier in bromocresol purple peptone broth (containing 0.5% sodium thiosulfate) at 56 for 7 days. Put the unsterilized carrier in PBS, dilute it. then inoculate on flat dish to make viable cell count. cultivate at 56 for 72h. 2.3 Results ( see Table l) Under the strengthened-cycle sterilization method (operating time: 89min. vacuum time: 15min, liquid injection volume:3ml. diffusion time: 2lmin. sterilization time: 13min). the low-temperature plasma sterilizer could kill Bacillus Stearothermophilus through twice successive sterilization. Table l. Result of On-Site Simulation Test Apparatus Parameter Test Test Positive Operating Pump Liquid Injection Diffusion Sterilization No. Date Control Volume(ml) Detection Result Growth Growth Growth No Growth No Growth No Growth No Growth No Growth No Growth # Liquid Injection Volume: The amount of hydrogen peroxide necessary for a single sterilization. The data after Nov.24 are parameters for adjusted whole sterilization process. 3 Clinical Materials Sterilization Test 3.1 Materials & Methods Neutralizer: 0.5% sodium thiosulfate M PBS. nutrient agar, nutrient broth. sterile normal saline Clinical materials: General operation apparatus (hemo-static forceps and materials-holding forceps of various types.10 pairs/set), peritoneoscopy (complete set. 10 materials/set). electrotome (including wire), drug-injection plastic tube. And Latex membrane. Wash all above materials. dry, and then pack for later sterilization. 3.2 Methods Wash routinely operation apparatus and electrotome (including wire), mop up, make double-cycle sterilization with50% hydrogen peroxide plasma sterilizer. smear repeatedly on materials ( from handle end to wire end in case of electrotome in purification operating bench with sterile cotton swab soaked with 0.5% sodium thiosulfate nutrient broth. shear off the part touched by hand, then put int0 0.5% sodium thiosulfate

4 nutrient broth. and cultivate at 37 for 7 days. Both peritoneoscope handling method and sampling method are same as above. For the materials with cavity, first rinse with 5ml sterile physiological saline. then inoculate the rinse solution in 0.5% sodium thiosulfate nutrient broth, and cultivate at 370C for 7 days. Wash routinely drug-injecting plastic tube and Latex membrane, mop up, make double-cycle sterilization with 50% hydrogen peroxide plasma sterilizer. shear one section with sterile scissors, put int0 0.5% sodium thiosulfate nutrient broth. and cultivate at 37 for 7 days. 3.3 Results (see Table 2, Table 3, and Table 4) Under the short -cycle sterilization method injection volume: 2ml. diffusion time: 9 ~ 14min, sterilization time: 2 ~ 4min), the drug-injecting tube, Latex membrane. and electrotome (including wire) were detected as free of bacteria after twice successive sterilization. Under the long-cycle sterilization method ( operating time: 71 ~ 79min, vacuum time: 14 ~ 16min, liquid injection volume:3ml. diffusion time: 16min. sterilization time: 9~12min), both operating apparatus and peritoneoscope were detected as free of bacteria after twice successive sterilization. (operating time: 36~51min. vacuum time: 4~8min, liquid Table 2 Result of Clinical Materials Sterilization Test Test Test Test Material Apparatus Parameter Detection No. Date Operating Pump Liquid Injection Volume(ml) Diffusion Sterilization Result Drug-injection tube No Growth Drug-injection tube No Growth Drug-injection tube No Growth Drug-injection tube No Growth Drug-injection tube No Growth Latex membrane No Growth Latex membrane No Growth Latex membrane No Growth Latex membrane No Growth Latex membrane No Growth Electrotome No Growth Electrotome No Growth Electrotome No Growth Electrotome No Growth Electrotome No Growth # Liquid Injection Volume: The amount of hydrogen peroxide necessary for 'a single sterilization. Electrotome includes its wire. 4 0rganic Substance Influence Test 4.1 Materials Neutralizer: 0.5% sodium thiosulfate M PBS. nutrient agar, nutrient broth Organic substance: 5% and 10% calf serum.

5 4.1.4 Test bacteria: Clinical drug-resistant bacteria strains( Staphylococcus aureus, Staphylococcus epidermidis, Fecal enterococcus. E coli. Enterobacter cloacae. Klebsiella pneumonia, and Stenotrophomonas maltoppilia ) 4.1.5φ1mm (inner diameter)x1000mm (long) PTFE hose, φ0.7mmix40mm steel needle,φ2mmx40mm PTFE hose ( as the casing at junction ). Plasma. Science &- Technology, V01.8. No.4. July 2006 Table 3 Result of Operation Apparatus Sterilization Test Test Test Apparatus Parameter Detection Result No. Date Operating Pump Liquid Injection Volume(ml) Diffusion Sterilization Growth No Growth Growth Growth No Growth No Growth Growth Growth No Growth No Growth No Growth No Growth No Growth No Growth No Growth # Liquid Injection Volume: The amount of hydrogen peroxide necessary for 'a single sterilization. The data after Nov.24 are parameters for adjusted whole sterilization process. Table 4 Result of Peritoneoscope Sterilization Test Test Test Apparatus Parameter Detection No. Date Operating Pump Liquid Injection Diffusion Sterilization Result Volume(ml) Growth No Growth Growth Growth No Growth No Growth No Growth No Growth No Growth # Liquid Injection Volume: The amount of hydrogen peroxide necessary for 'a single sterilization.

6 The data after Nov.24 are parameters for adjusted whole sterilization process. There was sediment in rinse solution in metal tube cavity of peritoneocsope, and thus it was not washed up. 4.2 Methods Preparation of organic substances Add calf serum into 0.03M PBS to prepare the PBS containing 5% and 10% calf serum respectively, prepare the cultivated test bacteria into 109CFU/ml bacterial suspension with the above PBS. Soakφ0.7mmx40mm steel needle in the bacterial suspension for 2h,take it out, and dry for later use. Incubate at 37 for 30min. Bacteria contamination and sterilization Place the bacteria-contaminated steel needle between the junction of two φ1mmx1000mm PTFE hoses. and seal with casing the inter-face of' two hoses. Pack the sealed hose into a special paper bag, seal the hag, put in a sterilization cabinet. and make double-cycle sterilization with 50% hydrogen peroxide. plasma(operating time 89min. vacuum LI Si et al. : Observation on Clinical Steri[ization Effectiveness of CASP-80 Low-Temperature time:15min, liquid injection volume: 3ml. diffusion time: 2lmin.sterilization time: 13min ). Detection Take out both sterilized and unsterilized carriers in hose. cultivate the sterilized carriers in 0.5% sodium thiosulfate nutrient broth at 37 for 7 days. Place the unsterilized carrier in PBS. dilute it. then inoculate on a flat dish to make a viable cell count. and cultivate at 37 for 48h. 4.3 Results ( see Table 5) Both 5%, and 10% organic substances (calf serum) could influence the sterilization effectiveness of CASP-80 low-temperature plasma sterilizer. Table 5 Result of Organic Substance Influence Test Test Bacteria Serum Concentration Amount of Recovered Bacteria Result Staphylococcus aureus 5% /5 10% /5 Staphylococcus epidermidis 5% /5 10% /5 Fecal enterococcus 5% /5 10% /5 E coli 5% /5 10% /5 Enterobacter cloacae 5% /5 10% /5 Klebsiella pneumonia 5% /5 10% /5 Stenotrophomonas 5% /5 maltoppilia 10% /5 *Number of positive bacteria/total number of test bacteria 5 Summary The plasma is a state of high activity and high energy [7. 8]. As the adjuvant of plasma sterilizer. 50% H is a strong oxidant which ionizes into plasma state in plasma sterilizer. attacks microbial lipid membrane. DNA and other important cells through active hydroxyl free radical. and thus achieves the sterilization. The plasma sterilizer

7 has a rapid sterilization speed of around l hour. The residual substances of sterilization are water and oxygen, are not left over at all. do not contaminate the environment. and form a safer medical environment. During the sterilization through plasma sterilizer, a long-cycle sterilization must be adopted to sterilize the body cavity(such as endoscope) due to the influence of permeability ( Table 4). In most modern researches. some disinfectant gases were added into plasma body cavity to greatly strengthen the sterilization effectiveness of plasma sterilizer [2.9-12]. The plasma sterilizer is one of main advances in sterilization apparatus in recent years. As proven by the results of extensive researches. the plasma sterilizer had very strong effects of killing the microorganisms, but could not be directly applied for the apparatus contaminated with blood. sodium chloride. and especially organic substances. As shown in Table 5. Both 5% and 10% organic substances (calf serum) could significantly influence the sterilization effectiveness of CASP-80 low-temperature plasma sterilizer possibly because the. Organic substance forms a protective layer on the surface of microorganism to prevent the contact with microorganism or delay its function. In most cases, the clinically used endoscope apparatus could be contaminated with organic substances (such as patient's saliva and blood). which would inevitably weaken the sterilization effectiveness of some disinfectants without washing or with incomplete washing. Thus. it is particularly important to completely clean up the sterilization apparatus before the sterilization. References I Brown SA, Merritt K, Woods TO. et al. Biomed Instrum Technol :23 2 0kpara-Hof'mann J. Knoll M. Durr M. et al. J Hosp Infect, :280 3 Lerouge S, Cuignot C, Tabrizian M. et al. J Biomed Mater Res :774 4 Rutala WA. Weber DJ. Emerg Infect Dis, :348 5 Kanemitsu K. Imasaka T. Ishikawa S. et al. Infect Control Hosp Fpidemiol :486 6 Mau T. Hartmann V. Burmeistcr J. et al. PDA J Pharm Sci Technol :130 7 Moisan M. Barbeau J. Moreau S. et al. Int J Pharm :1 8 Purevdorj D, Igura N, Ariyada O, et al. Lett Appl Microbiol :31 9 Selkon JB. J Hosp Infect : Ayliffe G.J. J Hosp Infect, 2000,45: Mosley GA, Gillis JR. Krushefski G. PDA J Pharm Sci Technol :64 12 Lerouge S, Tabrizian M. Wertheimer MR. et al. Biomed Mater Eng, 2002,12:3 ( Manusvript received 16 May 2006) addressof Corresponding author: ydmeng@ipp.ac.cn