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EtO Resistance of P. Domesticum
Originally Published MDDI March 2004Sterile Processing
March 1, 2004
9 Min Read
Originally Published MDDI March 2004
Medical products contaminated with the sclerotia of P. domesticum may not be sterile after an EtO cycle based on B. atrophaeus spore kill.
Steven G. Richter
Figure 1. Pyronema domesticum wild-type asci containing eight ascospores (400x).
The effectiveness of medical supply sterilization is clearly an important issue. Due in part to the diminishing efficacy of antibiotics, preventing the spreading of disease is of primary importance to the preservation of human health. All medical supplies that come in contact with the bodily fluids of patients and staff must be sterilized to ensure that they are free of infectious agents.
A number of methods, including radiation, steam, and ethylene oxide (EtO) gas, are currently used for sterilizing medical products. EtO gas is especially useful for materials that are unable to withstand the processing effects of high temperatures and radiation. Sterilization using EtO gas is a comprehensive procedure requiring rigid control of temperature, humidity, vacuum and positive pressure, and gas concentration.1 It is estimated that 45% of medical devices manufactured in the United States are sterilized using EtO gas.2 The validation process for EtO sterilization involves the use of product inoculated with biological indicators such as Bacillus atrophaeus (formerly B. subtilis var. niger) spore preparations.3, 4
Sterility is generally defined as freedom from viable microorganisms. However, to expect absolute freedom from these entities is unrealistic. Regardless of the method used for killing a microbial population, the death of that population occurs exponentially. Sterilization, therefore, is a probability function. The time or dose of a sterilizing agent required to effectively sterilize a product is stated as a D10-value, which represents the time or dose of sterilizing agent needed to lower the surviving fraction to 10% of the original population. Several methods are available for calculating D10-values, including the fraction negative techniques of Stumbo, Murphy, and Cochran (used in this study); the Spearman-Karber equation; and the Weibull distribution.5
In 1993, a manufacturer of sterile laparotomy sponges announced a voluntary recall of the sponges because of fungal contamination.6 In April 1994, FDA found that the prevalent organism, isolated from various lots of manufacturers' laparotomy sponges, was a mold identified as Pyronema domesticum.7 FDA found that several companies that were producing sponges made of cotton purchased from a supplier in China had been sterilizing with EtO gas. All were experiencing problems killing P. domesticum. 6, 8
Figure 2. Pyronema domesticum ATCC Strain 14881 apothecium from Claussen's Agar (100x).
P. domesticum is a fungus belonging to Division Ascomycota. Its sexual reproductive bodies are known as ascospores (see Figure 1), and these are produced in structures called apothecia (see Figure 2). P. domesticum also produces long-term survival mechanisms known as sclerotia (see Figure 3). Sclerotia are somatic structures designed to withstand unfavorable conditions. They are sometimes produced by ascomycetes that produce apothecia.9 Sclerotia are composed of hard masses of hyphae that germinate when favorable conditions arise, and a fruiting body or mycelium results.10 The ascospores and sclerotia of P. domesticum have been shown to be highly resistant to radiation.4 Previous studies suggest that P. domesticum is also resistant to sterilization using EtO gas, but no data have been published respecting the EtO resistance of the ascospores and sclerotia.
The study presented in this article was carried out to determine the EtO resistance of the sclerotia of P. domesticum.
Two strains of P. domesticum (deposited in the American Type Culture Collection by Elizabeth Moore-Landecker) were purchased and cultured using standard procedures. The fungal cultures were encouraged to produce sclerotia, and discs bearing sclerotia were prepared as described in a previous study (see Figure 3).4 The discs were exposed to EtO gas in a biological indicator evaluation resistometer (BIER) chamber, and all surviving sclerotia were counted. The results were used to calculate D10-values for the sclerotia of P. domesticum.
D10-values were calculated using the equation D = dose/(log N0 – log Nt) or D = dose/[log(N0/Nt)], which assumes exponential inactivation.5 Here N0 is the number of sclerotia exposed to EtO gas at time zero. Nt is the number of surviving sclerotia after exposure to EtO gas.
Results and Discussion
As shown in Figure 4, D10-value experiments revealed that P. domesticum sclerotia are resistant to EtO gas by a factor of 10 greater than B. atrophaeus spore preparations. The current study shows that at 600 mg/L EtO, the combined strain average D10-value was 33.9 minutes. It is important to note that FDA accepts the use of B. atrophaeus as a biological indicator for EtO sterilization and a D-value of 3 minutes at the same gas concentration.3 ISO 11135, the international standard for ensuring EtO sterilization of medical devices, promotes the use of biological indicators for this purpose.8
Figure 3. Pyronema domesticum ATCC Strain 32030 sclerotia used for ethylene oxide D10-value studies (40x).
B. atrophaeus spore strips are used as biological indicators by the majority of medical device manufacturers that use EtO gas for sterilization of their products.8 Based on the findings of these experiments, medical devices that are contaminated with the sclerotia of P. domesticum may not be sterile after a cycle that is based on B. atrophaeus spore kill.
Further studies regarding P. domesticum will focus on the EtO resistance of the ascospores of this organism. Previous research has shown that the ascospores of P. domesticum are highly resistant to radiation sterilization and that they are substantially more resistant than the sclerotia.4 Bacillus pumilus spores have been used in the past as biological indicators for validating the radiation sterilization process. Figure 5 indicates that the D10-value for B. pumilus is 1.7 kGy compared with a D10-value of 2.8 kGy for the ascospores of P. domesticum.11 It is possible that the ascospores of P. domesticum are also more resistant to EtO sterilization. Further research into this issue is imperative.
Figure 4. P. domesticum sclerotia are resistant to EtO gas by a
Microorganism resistance to EtO is clearly an important concern for the medical device industry. This study has demonstrated that P. domesticum is a highly EtO resistant organism. This organism has been found on medical devices containing cotton imported from China. These findings suggest that analysis of these products for this organism is crucial. Such analysis is essential for manufacturers to declare sterility. It is important to note, however, that bioburden and sterility testing as described in ISO 11737 may not address the use of optimum culture conditions for P. domesticum.
This organism may require a range of incubation temperatures, increased incubation periods, and varied media.4 A common method for discovering the presence of resistant organisms is presented in ISO 11135, which states that sublethal EtO cycles must be run in order to determine whether the biological indicator is more resistant than the natural bioburden. This procedure was developed to identify EtO resistant organisms such as P. domesticum.
Bioburden testing is an essential step in the manufacture of sterile medical devices. Medical product sterilization standards should be revised so that P. domesticum is routinely included in bioburden testing, especially when medical products are manufactured using foreign cotton. Bioburden test data are crucial in determining the quality of a finished product.
Figure 5. The D10-value for B. pumilus is 1.7 kGy. The ascospores of P. domesticum, by comparison, have a D10-value of 2.8 kGy. Click to enlarge.
FDA requires that medical device manufacturers meet a sterility assurance level (SAL) of 10–6 for all saleable sterile medical devices (10–3 for devices contacting intact skin only).13 Microbial inactivation during a sterilization process is described by an exponential function, and an SAL of 10–6 means that the probability of a single viable microorganism being present on a given product is one in one million after the sterilization process has been completed. A valid SAL must be documented, and SAL documentation requires a thorough knowledge of the product bioburden.14
The Code of Federal Regulations requires that medical device manufacturers develop a written procedure for bioburden testing that addresses the possibility of contamination by resistant organisms such as P. domesticum.15 The quality system regulation provides guidelines for proper manufacturing of medical devices where bioburden must be managed throughout the manufacturing process.
It is clearly essential that the medical device industry treat bioburden monitoring as a critical component of the manufacturing process. The assistance of qualified laboratories with experience in bioburden analyses and sterilization practices may be an effective means of achieving this goal.
01. “Sterilization and Sterility Assurance of Compendial Articles,” Section 1211, The United States Pharmacopoeia/The National Formulary, USP 25, (2002): 2251–2252.
02. J Masefield, “Advances Made in Cobalt-60 Gamma Sterilization,” Sterilization of Medical Products, vol II, (Montreal: Multiscience Publications, 1981).
03. Premarket Notifications [510(k)] for Biological Indicators Intended to Monitor Sterilizers Used in Health Care Facilities; Draft Guidance for Industry and FDA Reviewers, Tables 2 and 3, Food and Drug Administration, Available from Internet: www.fda.
04. S Richter and J Barnard, “The Radiation Resistance of Pyronema domesticum,” Journal of Industrial Micro Biotechnology, vol 29, (2002): 51–54.
05. SS Block, “Sterilization and Preservation by Ionizing Irradiation,” in Disinfection, Sterilization, and Preservation, (Philadelphia: Lea & Febiger, 1991), 571.
06. Recall of Laparotomy Sponges, Office of Surveillance and Biometrics, CDRH, Food and Drug Administration, 1993.
07. RM Johnson, Memo to All Device Manufacturers/Repackers Using Cotton. Office of Compliance, Center for Devices and Radiological Health, Food and Drug Administration, Rockville, MD, April 22, 1994.
08. Medical Devices—Validation and Routine Control of Ethylene Oxide Sterilization, ISO 11135, International Organization for Standardization, 1994.
09. E Moore-Landecker, Fundamentals of the Fungi, 4th ed., (Englewood Cliffs, NJ: Prentice-Hall, 1996).
10. B Kendrick, The Fifth Kingdom, 2nd ed., (Sidney, BC, Canada: Mycologue Publications, 1992).
11. M Burt and FJ Ley, “Studies of the Dose Requirement for the Radiation Sterilization of Medical Equipment,” Applied Bacteriology 26, (1963): 484.
12. Sterilization of Medical Devices—Microbiological Methods, ISO 11737, International Organization for Standardization, 1994.
13. Updated 510(k) Sterility Review Guidance K90-1; Guidance for Industry and FDA, U.S. Department of Health and Human Services, FDA, CDRH, Office of Device Evaluation, Aug. 30, 2002. Available from Internet: www.fda.gov/cdrh/ode/guidance/361.pdf.
14. SG Richter, “Product Contamination Control; A Practical Approach to Bioburden Testing,” Journal of Validation Technology, vol 5, no. 4, (1999): 333.
15. “Quality System Regulation; Production and Process Controls,” Title 21 Code of Federal Regulations, Part 820, 2002 ed. Available from Internet: www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/
The author is grateful to Elizabeth Moore-Landecker (Rowan College of New Jersey) for information on the ontogeny of Pyronema domesticum; also to Robin Richards-Quinteros for her work on this paper and on a previous paper, “The Radiation Resistance of Pyronema domesticum.” n
Steven G. Richter is president of MicroTest Laboratories (Agawam, MA).
Copyright ©2004 Medical Device & Diagnostic Industry
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