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The authors provide a review of test methodology and standards, including current industry and regulatory proposals, for biological indicator growout times.
A biological indicator is defined in the International Organization for Standardization (ISO) 11139 terminology document as: "a test system containing viable microorganisms providing a defined resistance to a specified sterilization process" (1). Biological indicators (BIs) are used by medical-device manufacturers, pharmaceutical manufacturers, and healthcare institutions to provide measured response of the efficacy of sterilization processes; the sterility of medical items cannot be inspected or tested in a practical manner. BIs use known quantities of micro-organisms with a high degree of resistance to specific sterilization processes to generate quantitative information regarding efficacy of the processes.
The presence of viable micro-organisms in or on a BI makes this form of sterilization indicator an appropriate tool to evaluate the sterilization process. BIs are unique in comparison with chemical indicators and physical monitors, which are also valuable and provide immediate or real-time information respectively. The latter monitors, however, do not directly provide a biological result.
That said, the use of viable micro-organisms requires a post-exposure incubation period to allow for growth of any test organisms that may have survived the sterilization process. This time delay in obtaining feedback from the sterilization cycle is often viewed as a major limitation to the use of BIs, particularly in applications for routine monitoring.
Viable micro-organisms, by nature, require time under suitable conditions of temperature, oxygen tension, nutrients, water, and so forth, to multiply and subsequently create a visual indication of their viability. The result of viability is typically demonstrated by turbidity in a broth culture medium, colonies on an agar surface, or a pH indicator or other metabolic-derived color changes that signify microbial metabolism and growth. A BI that is positive at any time during the predetermined incubation period that growth is detected is a straightforward result (i.e. a positive is a positive regardless of the incubation time). However, a negative BI result is ambiguous relative to the length of incubation. Because today's BIs use well-characterized bacterial endospores, all of the necessary conditions for germination and growth are established. Additionally, the performance requirements of BIs have been well characterized in the ISO 11138 BI Standards series (2). The only BI parameter that is not universally accepted to date is the length of the incubation time necessary to establish viability of BI organisms.
The release of medical items as "sterile" in both industry and in healthcare facilities is commonly based on negative BI monitoring results. The time from completion of the sterilization process to release of goods for distribution or patient use can have major economic and patient care implications. The incubation time at which a BI can be declared negative is therefore a significant issue and is to some extent complicated by the fact that any scientific conclusion based on a negative result is always open to debate.
The current ISO 14161:2009 standard, which provides guidance for users of BIs, recommends an incubation period of 7 days for established sterilization processes, such as ethylene oxide and moist heat, and 14 days for nonstandard or new sterilization processes (3). This guidance is consistent with the requirements of the BI manufacturer's standard ISO 11138-1:2006, but the 11138-1 document also specifies that the BI incubation time and temperature be validated (4). Additional text in ISO 14161:2009 regarding BI incubation time (12.3.3) states that:
Neither ISO BI document provides information on test methodology for validating BI incubation time. This lack of detail has led to confusing and nonuniform requirements for RIT documentation that have historically been applied to medical device and pharmaceutical manufacturers throughout the world.
The historical perspective
Prior to the mid 1980s, BIs were incubated for 7 days to obtain final results. However, it was commonly observed that BIs, particularly those used in moist-heat sterilization processes, rarely demonstrated positive results after the third day of incubation (5). Several studies confirmed these observations and industry began to petition FDA to allow the use of shorter incubation times for BIs (6, 7). In 1985, FDA adopted an interim position allowing for an incubation time of 5 days as the final BI readout time (8).
On Jan. 1, 1986, the agency's Center for Devices and Radiological Health (CDRH) published the FDA Guide for Validation of Biological Indicator Incubation Time (9). This document was initially intended for manufacturers of BIs as a guide for documenting incubation times of less than 7 days for their products. The method consists of a statistically based sampling plan requiring a minimum of 100 units from each of 3 different product lots, which implied three different spore crops. The sample sets are then exposed to a sublethal cycle designed to produce 30–80% positives after 7 days incubation. Using the number of positive BIs after 7 days incubation as the reference (or denominator in the calculation), the greatest number of days of incubation required to obtain greater than 97% of the positive BIs in any of the partial cycles (numerator data) is the minimum incubation time allowed per this method.
The CDRH guide may have initially been intended for BI manufacturers, but it soon became the default requirement for medical-device manufacturers relative to less than 7-day BI release of their sterilized medical products. Around the same time as the FDA guide was issued, the Association for the Advancement of Medical Instrumentation (AAMI) published its first BI standards through the American National Standards Institute (ANSI). ANSI/AAMI ST19: 1986 and ANSI/AAMI ST21: 1986 characterized BIs for use in ethylene oxide and moist heat sterilization processes respectively in health care facilities (10, 11). However, the only guidance regarding BI incubation time was to follow the BI manufacturers' instructions. A recommendation of 7 days incubation for BIs used in moist heat, ethylene oxide, and dry-heat sterilization processes first appeared in the United States Pharmacopoeia (USP) in 1990 and has remained unchanged per the recent 2011 USP edition (12, 13). Similar to the ISO and AAMI BI documents, the USP contains no information on test methodology for BI incubation time determination.
The initial version of ISO 11138-1, general requirements for BIs used in the sterilization of medical products, was issued in October 1994 (14). The European Committee for Standardization (CEN) published a similar document, EN 866-1, in 1997 (15). Both documents included requirements for general production, testing, labeling, and performance requirements for BIs used in healthcare sterilization processes, however, neither document contained information on BI incubation time other than to follow "methods and conditions prescribed by the (BI) manufacturer." A nearly identical version of ISO 11138-1, ANSI/AAMI ST59, was published in 1999; it also provided no direction regarding validation of BI incubation time (16).
During the mid-1990s, ISO initiated work to develop a BI user guidance document designated as ISO 14161. In the interest of harmonization, AAMI elected to parallel ballot this document, meaning that it would be adopted verbatim as an ANSI/AAMI/ISO standard following publication by ISO; the new document would also replace the ANSI/AAMI ST34:1991 standard for BI user guidance (17). There was consensus agreement by the ISO/Technical Committee (TC) 198 BI Working Group 4, and the AAMI BI Working Group 4 that methodology for determination of BI incubation time should be addressed in the 14161 document. Accordingly, early versions of ISO 14161 included an informative annex (Annex E) containing the 1986 CDRH Guide for validation of BI incubation time because it was the only method available for this determination.
Subsequent ISO and AAMI BI working group discussions regarding the CDRH guide raised significant issues regarding the utility of this methodology. Input from BI working group members indicated that it was difficult to obtain reproducible results with the CDRH method, and that it was also cumbersome to consistently obtain between 30 and 80% BI positives after 7 days incubation. As a result of continuing discussions, an alternative method for validation of BI incubation time was introduced to the ISO and AAMI BI working groups. The newer methodology was based on a commercial BI manufacturer's protocol and represented a statistically consistent but less conservative approach compared with the CDRH method for determining BI incubation times. In contrast to the CDRH method requirement of > 97% correlation between the 7 day incubation results and the RIT, the alternative method required a correlation of 95% but also used a larger sample size of 200 units per lot. Comments submitted to the ISO/TC 198 WG4 and AAMI WG4 on the alternative method were that it favored BI producers and that it was unproven for widespread use in industry.
As a result of these discussions, a request was made at the June 1997 AAMI BI WG meeting that a statistical analysis be performed comparing the operating characteristics of the two methods in question. The CDRH method was determined to be restrictive in terms of the probability for acceptance of shortened incubation times having a Rejectable Quality Level (RQL; protects consumer) of 0.036 at = β 0.1 and an Acceptable Quality Level (AQL; protects producer) of 0.005 at α = 0.1. The alternative method was shown to have a significantly higher RQL and AQL and provide a higher probability for acceptance of reduced incubation time data. Based on a statistical simulation, the CDRH guide was shown to have a probability of acceptance of 30 % when using a theoretical "true growth readout" of > 97%, whereas the alternative method would accept this same theoretical input virtually 100% of the time (18).
Ultimately, neither method was considered to be satisfactory by the ISO or AAMI BI working groups. A joint agreement was reached that Annex E should be removed from the 14161 draft so that the standard could move forward for publication. These decisions, however, also included agreement that the subject was important relative to BI use and, therefore, a New Work Item Proposal (NWIP) would be initiated to develop an ISO Technical Specification (TS) that would contain methodology for validation of BI incubation time. This document, designated as ISO TS 16342, was developed in early 2000 and presented to the ISO BI WG later that year (19). The ISO TS 16342 draft document contained the CDRH method and the proposed industry alternate method; these two methods were intended to be used as a starting point for development of a new methodology. It was anticipated that under the auspices of the ISO and AAMI BI working groups, a hybrid method acceptable to both regulators and users could be developed based on the two statistically diverse approaches. After years of discussion, but at a low priority due to revision of the ISO 11138 series standards, there was little movement and TS 16342 was subsequently taken off of ISO's agenda due to the lack of progress.
Although the issue of BI incubation time was still considered relevant, there was little formal activity on the subject within the working groups until 2005, when work began to revise ISO 14161:2000. The goal of revision was to align the standard with the newly revised ISO 11138 BI standard series. Comments submitted on the ISO 14161 revision included requests from five different member countries that were related to development of methodology for validating BI incubation time. At the 2007 general meeting of ISO/TC 198 working groups in Dublin, Ireland, the BI working group agreed that the dormant NWIP to address this issue (i.e., TS 16342) should be re-instated as an active project. The working group also requested that any proposed methodology be supported by actual data and published in a peer-reviewed scientific journal to support technical credibility. Due to the relatively long time periods between ISO/TC198 general meetings, the commitment was made by AAMI members of the ISO BI Working Group 4 to take the lead on this project. Following discussions at the subsequent AAMI Working Group 4 BI meetings, an ad hoc committee was created in 2008 to identify a plan for gathering data and develop a new test protocol for RIT determination.
Status of ISO/AAMI Working Group 4 activity
In 2009, in conjunction with the AAMI BI Working Group ad hoc committee, a study was initiated to examine growout times of Geobacillus stearothermophilus BIs exposed at 121 °C in moist-heat sterilization cycles. The data were generated using a technology that continuously monitors incubated BIs and automatically records growth-positive results (20). The system facilitated accumulation of approximately 4000 data points. Per the ISO BI Working Group directive, the study was published in 2010 and is the first in a series of planned publications designed to provide a data-based scientific rationale for a proposed RIT test methodology. Observations and conclusions from this first study include (21):
An additional study examined growout times for Bacillus atrophaeus BIs exposed to ethylene oxide (EO) and chlorine dioxide gas-sterilization processes, and Geobacillus stearothermophilus BIs exposed to hydrogen-peroxide vapor sterilization process and moist-heat processes at 132–135 °C. The resulting data were consistent with the observations and conclusions from the initial study. A final proposed test method based on the aggregated data is planned to be submitted by the AAMI WG4 ad hoc committee to the ISO and AAMI BI working groups for review, discussion, and possible modification for inclusion into the ISO TS 16342 draft document. The target date for submission is fourth quarter 2012.
Philip M. Schneider is a senior consultant with LexaMed, 705 Front Street, Toledo, OH 43604, email@example.com and is Convener of ISO/TC 198 WG4 Biological Indicators. John R. Gillis is owner of J.R. Gillis & Associates, 2303 Nelson Road, Bozeman, MT 59715, firstname.lastname@example.org and is an Expert Member of ISO/TC198 WG4 Biological Indicators.
1. ISO TS 11139:2006 (ISO, Geneva, 2006).
2. ISO 11138 Series (ISO, Geneva, 2006).
3. I SO 14161:2009 (ISO, Geneva, 2009).
4. ISO 11138-1:2006 (ISO, Geneva, 2006)
5. P.J. McCormick, Memorandum presented to AAMI BI Working Group, 2001.
6. R.C. Kralovic, Pharm. Tech. 6, (11) 100–103 (1982).
7. H.W. Winckels and P.G.F. Boumans, proceedings of the EUCOMED Conference: Sterilization Validation of Medical Devices and Surgical Products (Copenhagen, 1984).
8. P.J. McCormick, Memorandum to Lois Jones, AAMI Biological Indicator Incubation Time Validation Task Group, June 28, 1999.
9. FDA CDRH, Guide for Validation of Biological Indicator Incubation Time (June 5, 1985).
10. ANSI/AAMI ST19:1986 (AAMI, Arlington, VA, 1986).
11. ANSI/AAMI ST21:1986 (AAMI, Arlington, VA, 1986).
12. USP 22–NF 17 General Chapter <1211>, "Sterilization and Sterility Assurance of Compendial Articles," 1990.
13. USP 34–NF 29 General Chapter <1211>, "Sterilization and Sterility Assurance of Compendial Articles," 2011.
14. ISO 11138-1: 1994 (ISO, Geneva, 1994).
15. CEN, EN 866-1: 1997 (CEN, Brussels, 1997).
16. ANSI/AAMI ST59: 1999 (AAMI, Arlington, VA, 1999).
17. ANSI/AAMI ST34: 1991 (AAMI, Arlington, VA, 1991).
18. H.F. Bushar, Memorandum presented to AAMI BI Working Group, 1997.
19. ISO TS CD 16342: 2000 (ISO, Geneva, 2000).
20. Smart-Read EZTest Biological Indicator Monitoring System, Mesa Labs, Bozeman, Montana.
21. J.R. Gillis et al., Pharm. Tech., 34 (1), online (2010).
22. H.O. Halverson and N.R. Ziegler, J. Bacteriology 25, 101–121 (1933).