Characterization of methods for determining sterilization efficacy and reuse efficiency of oxygen biosensor multiwell plates

High-throughput screening (HTS) assays based upon fluorometric detection of oxygen consumption in microtiter plates were primarily developed for applications in drug discovery and ecotoxicology but have recently been adopted for use in microbial community-level physiological profiling assays (CLPP). The widespread use of oxygen biosensor systems for CLPP applications has, however, been hindered by the relatively high cost of oxygen biosensor reagent systems and limited access to microplate fluorometer instrumentation platforms. The ability to recycle and reuse oxygen biosensor system plates would expand their utilization for CLPP assays and other research applications in microbial ecology. Here, the efficacy and cost effectiveness of multiple procedures for sterilization of Oxygen Biosensor System (OBS; BD Biosciences) plates for reuse was evaluated. OBS plates were sterilized using ethylene oxide, ultraviolet radiation, and bleach treatments, then evaluated for biosensor response and plate life-cycle performance. Of the sterilization methods tested, ethylene oxide sterilization was most effective based on its low cost, high sterilization efficacy, and minimal impact upon OBS plate response.     

Characterization of methods for determining sterilization efficacy and reuse efficiency of oxygen biosensor multiwell plates

High-throughput screening (HTS) assays based upon fluorometric detection of oxygen consumption in microtiter plates were primarily developed for applications in drug discovery and ecotoxicology but have recently been adopted for use in microbial community-level physiological profiling assays (CLPP). The widespread use of oxygen biosensor systems for CLPP applications has, however, been hindered by the relatively high cost of oxygen biosensor reagent systems and limited access to microplate fluorometer instrumentation platforms. The ability to recycle and reuse oxygen biosensor system plates would expand their utilization for CLPP assays and other research applications in microbial ecology. Here, the efficacy and cost effectiveness of multiple procedures for sterilization of Oxygen Biosensor System™ (OBS; BD Biosciences) plates for reuse was evaluated. OBS plates were sterilized using ethylene oxide, ultraviolet radiation, and bleach treatments, then evaluated for biosensor response and plate life-cycle performance. Of the sterilization methods tested, ethylene oxide sterilization was most effective based on its low cost, high sterilization efficacy, and minimal impact upon OBS plate response.     

Microbiological Aspects of Ethylene Oxide Sterilization: I. Experimental Apparatus and Methods

A specially built thermochemical death-rate apparatus is described which can be used to determine the resistance of microorganisms to ethylene oxide under controlled conditions. The apparatus was designed to provide instantaneous exposure of microorganisms to ethylene oxide and to eliminate variables that could result in errors when death kinetic reaction rates are calculated. The apparatus is used to obtain ethylene oxide resistance data which are useful in evaluating and developing sterilizing cycles for materials with known bacterial concentrations, as well as for calculating probability factors on which a given test condition can be expected to provide sterilization.     

不同消毒方法对生物化PET 人工韧带的生物力学影响

目的:探讨环氧乙烷、高压蒸汽、60Co照射三种消毒方法,对生物化PET人工韧带的生物力学影响,为选取合适的消毒灭菌方法提供实验依据。方法:以表面改性的聚对苯二甲酸乙二醇酯纤维为材料,制备生物化PET人工韧带,分别给予环氧乙烷熏蒸、高压蒸汽、60Co照射消毒处理,并设未作消毒处理的空白对照组,分别进行生物力学测试,对测量结果行统计学比较分析。结果:高压蒸汽消毒后,韧带所能承受的最大拉力及第一次破裂力降低,且与对照组间的差异均有统计学意义(P<0.05);60Co照射消毒后,韧带所能承受的最大拉力降低,与对照组间的差异有统计学意义(P<0.05),但第一次破裂力的差异不明显(P>0.05);环氧乙烷消毒处理后,最大拉力、第一次破裂力与对照组之间无明显差异(P>0.05);各组间断裂伸长率无明显差异(P>0.05)。结论:高压蒸汽消毒后韧带样品的生物力学性能降低比较明显;采用60Co射线消毒方法对产品的力学性能影响较小,但进一步的消毒方法改进可能更为理想;环氧乙烷消毒法可作为生物化PET人工韧带的有效消毒方法。     

Improved Sterilization of Sensitive Biomaterials with Supercritical Carbon Dioxide at Low Temperature

The development of bio-resorbable implant materials is rapidly going on. Sterilization of those materials is inevitable to assure the hygienic requirements for critical medical devices according to the medical device directive (MDD, 93/42/EG). Biopolymer-containing biomaterials are often highly sensitive towards classical sterilization procedures like steam, ethylene oxide treatment or gamma irradiation. Supercritical CO₂ (scCO₂) treatment is a promising strategy for the terminal sterilization of sensitive biomaterials at low temperature. In combination with low amounts of additives scCO₂ treatment effectively inactivates microorganisms including bacterial spores. We established a scCO₂ sterilization procedure under addition of 0.25% water, 0.15% hydrogen peroxide and 0.5% acetic anhydride. The procedure was successfully tested for the inactivation of a wide panel of microorganisms including endospores of different bacterial species, vegetative cells of gram positive and negative bacteria including mycobacteria, fungi including yeast, and bacteriophages. For robust testing of the sterilization effect with regard to later application of implant materials sterilization all microorganisms were embedded in alginate/agarose cylinders that were used as Process Challenge Devices (PCD). These PCD served as surrogate models for bioresorbable 3D scaffolds. Furthermore, the impact of scCO₂ sterilization on mechanical properties of polysaccharide-based hydrogels and collagen-based scaffolds was analyzed. The procedure was shown to be less compromising on mechanical and rheological properties compared to established low-temperature sterilization methods like gamma irradiation and ethylene oxide exposure as well as conventional steam sterilization. Cytocompatibility of alginate gels and scaffolds from mineralized collagen was compared after sterilization with ethylene oxide, gamma irradiation, steam sterilization and scCO₂ treatment. Human mesenchymal stem cell viability and proliferation were not compromised by scCO₂ treatment of these materials and scaffolds. We conclude that scCO₂ sterilization under addition of water, hydrogen peroxide and acetic anhydride is a very effective, gentle, non-cytotoxic and thus a promising alternative sterilization method especially for biomaterials.     

Increased frequency of sister chromatid exchange in persons occupationally exposed to ethylene oxide

Mutagenic effects of ethylene oxide have been demonstrated by short-term testing in vitro and in vivo in several organisms. Its oncogenic activity for man has been suspected and recently supported by experiments in mice. Exposure can occur during ethylene oxide gas sterilization of medical materials. We have tested effects on chromosomes by estimating sister chromatids exchange (SCE) frequency. Our results, reported here, show that exposure to this substance during work is followed by a very highly significant increase of SCE frequency as compared with controls. Thus, the SCE test may, under particular conditions, represent a reliable test for exposure to certain toxic agents.     

Resistance of Micro-organisms to Inactivation by Gaseous Ethylene Oxide

A simple method for the exposure of micro-organisms to ethylene oxide on membrane filters in a modified desiccator has been devised and used to study microbial resistance to the gaseous sterilant and the term ' R -value' is suggested to express this. The resistance of many known species and isolates has been assessed and compared. Several species of Bacillus were isolated from natural habitats and their spores were found to be more resistant than the strain of Bacillus subtilis var. niger (NCTC 10073) frequently used to monitor ethylene oxide sterilization. However, endospores of some bacterial species exhibited little resistance. Fungal spores and vegetative bacteria exhibited low resistance to the sterilant except after drying in organic material when they appeared more resistant than spores of B. subtilis var. niger. It was concluded that resistance to ethylene oxide did not correlate with resistance to heat, irradiation or other chemical disinfectants, or to the existence in the endospore form per se.     

Ethylene Oxide Resistance of Nondesiccated and Desiccated Spores of Bacillus subtilis var. niger Hermetically Sealed in Various Polymeric Films

The resistance to destruction of spores of Bacillus subtilis var. niger hermetically sealed in various polymeric films and exposed to ethylene oxide with and without relative humidity was determined. The effect of desiccation was also determined. The order of increased resistance to sterilization with regard to type of polymeric film was found to be: polyethylene equal to polyvinyl chloride, less than nylon, less than cellophane/polyethylene laminate, less than phenoxy, less than mylar/polyethylene laminate. Desiccated spores sealed in various polymeric films were much more resistant to ethylene oxide sterilization than nondesiccated spores. Relative humidity was an important factor in ethylene oxide sterilization with spores not sealed in polymeric films. However, with spores hermetically sealed in polyethylene, added relative humidity was an insignificant factor in the sterilization process.     

Impact of a bilingual mobile spay/neuter clinic in a U.S./Mexico border city

There are between 4 and 10 million dogs and cats killed annually in the United States. Although there are no accurate national estimates of the number of companion animals who are sterilized surgically. Approximately 26,000 companion animals are euthanized annually in El Paso County, Texas, located on the U.S./Mexico border. In an effort to determine if a readily available spay/neuter program would be cost effective and eventually help to lower the county's euthanasia rate, a mobile spay/neuter clinic began operation for a 5-month period in 2004, using a volunteer veterinarian and paid staff. Sterilizations performed totaled 1,108: 959 dogs (372 males and 587 females) and 149 cats (50 males and 99 females). The per companion animal sterilization cost of $15.13 ($27.83 had the veterinarian been paid) was considerably cheaper than the rate of $57 per companion animal achieved by a local voucher program contracting with private veterinarians to perform reduced-cost sterilizations.     

Family planning programmes in ten developing countries: cost effectiveness by mode of service delivery

The cost effectiveness of various modes of family planning service delivery based on the cost per couple-year of protection (CYP) is assessed using 1984 data for 63 projects in ten countries (three each in Africa and Asia, and four in Latin America). More than 4.8 million CYPs were provided through these projects during the year studied. Programmes with the highest volume of services delivered corresponded to lowest average costs: social marketing (2.8 million CYPs) and sterilization projects (960,000 CYPs) cost about $2 per CYP, on average; highest costs were for full service clinics and community-based distribution projects ($13-14 per CYP). Costs of clinics combined with community-based distribution services fell approximately midway between these two extremes.     

Diagnostic Accuracy and Cost-Effectiveness of Alternative Methods for Detection of Soil-Transmitted Helminths in a Post-Treatment Setting in Western Kenya

Objectives

This study evaluates the diagnostic accuracy and cost-effectiveness of the Kato-Katz and Mini-FLOTAC methods for detection of soil-transmitted helminths (STH) in a post-treatment setting in western Kenya. A cost analysis also explores the cost implications of collecting samples during school surveys when compared to household surveys.

Methods

Stool samples were collected from children (n = 652) attending 18 schools in Bungoma County and diagnosed by the Kato-Katz and Mini-FLOTAC coprological methods. Sensitivity and additional diagnostic performance measures were analyzed using Bayesian latent class modeling. Financial and economic costs were calculated for all survey and diagnostic activities, and cost per child tested, cost per case detected and cost per STH infection correctly classified were estimated. A sensitivity analysis was conducted to assess the impact of various survey parameters on cost estimates.

Results

Both diagnostic methods exhibited comparable sensitivity for detection of any STH species over single and consecutive day sampling: 52.0% for single day Kato-Katz; 49.1% for single-day Mini-FLOTAC; 76.9% for consecutive day Kato-Katz; and 74.1% for consecutive day Mini-FLOTAC. Diagnostic performance did not differ significantly between methods for the different STH species. Use of Kato-Katz with school-based sampling was the lowest cost scenario for cost per child tested ($10.14) and cost per case correctly classified ($12.84). Cost per case detected was lowest for Kato-Katz used in community-based sampling ($128.24). Sensitivity analysis revealed the cost of case detection for any STH decreased non-linearly as prevalence rates increased and was influenced by the number of samples collected.

Conclusions

The Kato-Katz method was comparable in diagnostic sensitivity to the Mini-FLOTAC method, but afforded greater cost-effectiveness. Future work is required to evaluate the cost-effectiveness of STH surveillance in different settings.     

Rapid Syphilis Testing Is Cost-Effective Even in Low-Prevalence Settings: The CISNE-PERU Experience

Studies have addressed cost-effectiveness of syphilis testing of pregnant women in high-prevalence settings. This study compares costs of rapid syphilis testing (RST) with laboratory-based rapid plasma reagin (RPR) tests in low-prevalence settings in Peru. The RST was introduced in a tertiary-level maternity hospital and in the Ventanilla Network of primary health centers, where syphilis prevalence is approximately 1%. The costs per woman tested and treated with RST at the hospital were $2.70 and $369 respectively compared with $3.60 and $740 for RPR. For the Ventanilla Network the costs per woman tested and treated with RST were $3.19 and $295 respectively compared with $5.55 and $1454 for RPR. The cost per DALY averted using RST was $46 vs. $109 for RPR. RST showed lower costs compared to the WHO standard costs per DALY ($64). Findings suggest syphilis screening with RST is cost-effective in low-prevalence settings.     

A Bacterial Spore Test Piece for the Control of Ethylene Oxide Sterilization

Aluminium foil strips carrying varying numbers of spores of the Camp Detrick strain of Bacillus subtilis dried from water, 90% (v/v) methanol, 5 and 20% (v/v) serum, nutrient broth and isotonic saline were examined for their possible use as test pieces for the control of sterilization by ethylene oxide. Methanolic suspensions of these spores were found to be stable on storage, and foils carrying spores dried from methanol were the most reproducible and stable of those tested. The susceptibility of the test piece system could be controlled by varying the numbers of spores on each foil and the numbers of foils used per test.     

Effectiveness of certain sterilizing agents for seedling incubation in radiothymidine

Failure of seed sterilization methods and breakdown in sterile technique can result in catabolism of thymidine by microorganisms during seedling incubation in radioisotope. The effectiveness of some sterilizing agents was monitored by microbiological methods and by thin layer chromatography for the presence of thymidine degradation products. Sodium hypochlorite and ethylene oxide each sterilized pea and sunflower seeds. Sodium hypochlorite failed to sterilize onion seeds while ethylene oxide was only occasionally effective and often retarded germination. Captan, commonly used before germination to prevent fungal blight of seedlings, did not reduce the bacterial flora of onion seeds.     

Radiation and Ethylene Oxide Terminal Sterilization Experiences with Drug Eluting Stent Products

Radiation and ethylene oxide terminal sterilization are the two most frequently used processes in the medical device industry to render product within the final sterile barrier package free from viable microorganisms. They are efficacious, safe, and efficient approaches to the manufacture of sterile product. Terminal sterilization is routinely applied to a wide variety of commodity healthcare products (drapes, gowns, etc.) and implantable medical devices (bare metal stents, heart valves, vessel closure devices, etc.) along with products used during implantation procedures (catheters, guidewires, etc.). Terminal sterilization is also routinely used for processing combination products where devices, drugs, and/or biologics are combined on a single product. High patient safety, robust standards, routine process controls, and low-cost manufacturing are appealing aspects of terminal sterilization. As the field of combination products continues to expand and evolve, opportunity exists to expand the application of terminal sterilization to new combination products. Material compatibility challenges must be overcome to realize these opportunities. This article introduces the reader to terminal sterilization concepts, technologies, and the related standards that span different industries (pharmaceutical, medical device, biopharmaceuticals, etc.) and provides guidance on the application of these technologies. Guidance and examples of the application of terminal sterilization are discussed using experiences with drug eluting stents and bioresorbable vascular restoration devices. The examples provide insight into selecting the sterilization method, developing the process around it, and finally qualifying/validating the product in preparation for regulatory approval and commercialization. Future activities, including new sterilization technologies, are briefly discussed.     

Sterilization with gaseous ethylene oxide: A review of chemical and physical factors

Although the basic parameters for ethylene oxide sterilization are established, it is sometimes difficult to attain in practice where the principal limiting factor is moisture availability. There are situations which can limit or enhance the dynamics of sterilization. Such factors, if overlooked, could upset experiments and lead to erroneous conclusions, or defeat the sterilization process entirety. Such are, namely: stratification effects, diffusion barriers, moisture-reducing effects, polymerization, and temperature distribution gradients.     

Comparison of European and US biological indicators for ethylene oxide sterilization

Summary Biological indicators (BIs) are used to monitor ethylene oxide (EO) gas sterilization processes for medical devices. Several European and United States BIs for EO sterilization were evaluated for resistance according to both United States Pharmacopeia (USP) XXI and United Kingdom's (UK) tests for D-values. US BIs areB. subtilis var. niger spores on paper strips or disc carriers while European BIs use aluminum strips, quartz sand, or cotton yarn. Numerous BIs per run and runs per lot, as well as 2–3 different lots of BIs from each manufacturer, were examined. Both British and US BIs met their respective label claims for rates of inactivation when tested against British and USP EO test parameters, respectively. However, Danish BIs, on cotton yarn or quartz sand, were not inactivated following USP specifications during the exposure dwell times tested (600 mg L^–1 EO, 54°C, 60% RH, 0–110 min). The Danish BIs will require further testing in order for us to determine if theirB. subtilis spores are unusually resistant to EO or if the spore carrier substrates protect the spores from the sterilizing gas. In conclusion, the British and American BIs for EO sterilization are equivalent in resistance despite differences in carrier substrate, recovery conditions, calculation methods for D-values, and the labeled sterilization conditions for use.     

Effects of sterilization treatments on some properties of alginate solutions and gels.

Aqueous sodium alginate solutions were subjected to various heat sterilization treatments. Sodium alginate powder was also treated by both gamma-irradiation and ethylene oxide sterilization. The effects of these treatments on the viscosities of sodium alginate solutions and both the diameter and strength of the beads formed in 0.1 M CaCl2 solutions were determined quantitatively. The viscosity of sodium alginate solutions and the gel strength of the calcium alginate beads decreased with increasing sterilization temperature while the bead diameters were found to increase. All these effects can be attributable to a reduction in the degree of polymerization of the alginate molecules as a result of the heat treatments. Ethylene oxide and gamma-irradiation treatments caused similar effects. Standard conditions for sterilization are necessary for comparative studies with alginate beads.     

Sorption Processes in Gas Sterilization in the Medical Sector

Sorption of ethylene oxide during and after gaseous sterilization is influenced by numerous factors. It was found that ethylene oxide desorption not only depends on material to be fumigated but also to a considerable degree on the wrapping material. Although polyethylene, polyamide (nylon), polytetrafluoroethylene (Teflon), silicone, aluminum, and glass beads contained no quantities of ethylene oxide detectable by gas chromatography after 72 h of aeration, residual amounts were definitely determined, even after 76 h of aeration in polypropylene, polystyrene, polyvinylchloride, paper products, and compound products of various plastics and paper mixtures. Desorption was, in all cases, found to be better when a mixture of ethylene oxide and methyl formate was used instead of pure ethylene oxide.     

Results of cytogenetic testing of workers exposed to ethylene oxide

The present work was undertaken to test cytogenetically workers exposed to Ethylene oxide (EO) aiming at detecting possible adverse effects of this agent to man. In the period 1983-1984, female workers of the Laboratory of Toxicology and the Production laboratory of ethylene oxide production in a chemical plant, and female workers in a sterilization centers in a medical facilities, were repeatedly examined. In 1984, also workers in ethylene oxide production in a chemical plant were tested. For each exposed group a control group was simultaneously examined, using cytogenetic analysis of peripheral blood lymphocytes. General examination of the health condition of exposed workers were organized in the framework of preventive examinations. The exposure level to ethylene oxide in the working environment was simultaneously monitored. The results have revealed an increase in chromosomal aberrations in lymphocytes of persons exposed professionally to EO at the given exposure levels. Smoking was associated with increased frequency of chromosomal aberrations in exposed workers; the difference as compared to non-smokers was however insignificant. By contrast, difference in chromosomal aberration frequency between smokers and non-smokers was significant in the control group.