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Performance Characterization and Comparison of Two Rapid Readout Biological Indicators for Use in Moist Heat Sterilization ProcessesP.M. Schneider
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Sterilization of medical items is central to the control and prevention of healthcare-associated infections (HAIs). Sterility, however, is a condition that cannot be visually or mechanically inspected nor tested by any practical means. Consequently, one of two possible strategies is used to ensure the effectiveness of sterilization cycles: validation of the sterilization process or routine monitoring with sterilization indicators.
Manufacturers of medical devices and pharmaceutical products validate their sterilization processes through exhaustive ‘up front’ testing to determine and document the capabilities of their respective processes. This knowledge and understanding of their sterilization processes then becomes part of a Quality System which contains policies and procedures covering virtually every aspect of product manufacturing. Strict adherence to these policies and procedures on a continual basis combined with the consistency of similar product loads creates a high probability of sterility for processed items. The second strategy for assurance of sterility utilizes recommended practice guidelines for the reprocessing of medical items in conjunction with routine monitoring of the sterilization process by means of various sterilization monitors. Routine monitoring is used predominately by healthcare facilities in that they do not have the resources necessary to validate their sterilization processes nor are the sterilizer loads in these facilities consistent on a day to day basis making validation even more improbable.
Routine monitoring of the sterilization process in healthcare facilities is advocated by a number of notable healthcare focused organizations including AAMI (Association for the Advancement of Medical Instrumentation), CDC (Centers for Disease Control and Prevention) and AORN (Association of periOperative Registered Nurses).1,2,3 These organizations recommend monitoring of sterilization cycles with a combination of physical (or mechanical) monitors, chemical indicators (CIs) and biological indicators (BIs). Each of these monitors is unique and has both advantages and limitations.
Physical monitors include gauges, LED readouts, charts and printouts and are usually an integral component of the sterilizer. They are the only method of monitoring that provides ‘real time’ information regarding the cycle. The readouts from these monitors, however, are generally produced by a sensor positioned at only one location within the sterilizer chamber (i.e., the drain in a steam sterilizer). Therefore these types of monitors provide no information regarding temperature or sterilant penetration inside a packaged item.
CIs consist of a thermo sensitive and/or chemical sensitive ink that is printed on a paper, plastic or foil substrate. They can be placed either outside or inside a package containing a medical item. CIs provide immediate information on some or all parameters of the sterilization cycle but unless the sterilization package has a clear plastic component, the package must be opened to determine the readout of the internal CI. The size and relative cost of this type of indicator allows for multiple placements throughout the sterilizer load. In contrast to physical monitors, CIs may detect errors in procedure relative to incorrect packaging or incorrect loading of the sterilizer.1,2 The primary limitation of CIs is that they are non-biological by design and therefore do not always reflect that the conditions in the sterilization cycle are sufficient to inactivate microorganisms.
BIs contain living microorganisms that have been shown to be highly resistant to the sterilization process in which they are used and as such provide an indication of the lethality, i.e., the microbial killing power, of the sterilization process. Since the bacterial spores generally used for BIs are both more resistant to the selected sterilization process and present in greater numbers than the microorganisms commonly found on medical items, inactivation of the BI provides a strong implication that the contaminating microorganisms have also been killed.4 While physical monitors and CIs contribute valuable information regarding the sterility of a processed load, BIs are recognized by most authorities as being closest to the ideal monitors of the sterilization process.2,5 The primary limitation
Introduction
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of BIs is that they require incubation before results can be obtained. This incubation time is usually 24–48 hours to obtain a pH color change indicating growth for Geobacillus stearothermophilus self-contained BIs used to monitor steam sterilization cycles.
In the early 1990’s, rapid readout BIs for the steam process were developed and commercialized which reduced the incubation time to 1-3 hours depending on the type of cycle used. For the steam process, this rapid readout system uses a Geobacillus stearothermophilus spore associated enzyme to reliably predict spore growth after the 1-3 hour incubation period.6 Growth is then evidenced by the production of acid metabolites produced by the Geobacillus stearothermophilus organisms upon further incubation. Since its introduction into the marketplace, these Attest™ Rapid Readout BIs have been used in health care facilities throughout the world for monitoring the steam sterilization process and ultimately to provide a criterion for the release of steam sterilized items for patient applications. Additionally, this product has been used for the validation and product release of medical devices in the industrial sector.7
Recently, a new rapid readout BI for monitoring the steam sterilization process has become commercially available. This newer product is very similar in design to the Attest™ Rapid Readout BI and is being marketed as having equivalent performance to the Attest™ product in monitoring of steam sterilization cycles. The objective of this study was to compare the performance of the 3M™ Attest™ 1292 Rapid Readout Biological Indicators with that of the newer TERRAGENE Bionova® BT220 Rapid Readout biological indicators in 121°C and 132°C steam sterilization cycles. The data generated in this study were then used to address the purported equivalency of the two products.
Overview
Side by side testing was performed with 3 different lots of 3M™ Attest™ 1292 Rapid Readout Biological Indicators (780 units) and 1 lot of TERRAGENE Bionova® BT220 Rapid Readout biological indicators (260 units). The testing was conducted by exposing the products at various times in both 121ºC gravity displacement cycles and 132ºC vacuum assisted cycles in steam resistometers (see resistometer description below). Following exposure, 3 hour fluorescent results and 24, 48 and 168 hour pH color change (growth) results were determined for each of the BIs. Any BI that showed a negative fluorescence reading at 3 hours but that demonstrated growth upon further incubation was considered to be a ‘fluorescent false negative’ (FFN) result.
Population counts were determined for each lot of product and this information was used in conjunction with data from those exposures producing fraction negative results at 121°C to calculate D121 values for each product lot. These experimentally determined population counts and D121 values were then compared to the respective BI manufacturers’ certification values.
Sterilizers
Joslyn Steam Resistometer meeting the requirements of ISO 184728
H&W Steam Resistometer, meeting the requirements of ISO 18472Note: A resistometer is a specially designed sterilizer with a small chamber and rigid instrumentation requirements to provide a high degree of control and reproducibility for the testing of sterilization indicators. ISO 18472 describes the performance specifications for resistometers (including steam).
Sterilizer racks
Metal racks designed for holding multiple self-contained BIs
Materials and Methods
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Biological Indicators
I. 3M™ Attest™ 1292 Rapid Readout Biological Indicator
• Self-contained BI requiring incubation @ 60 ± 2°C with 3 hour fluorescent readout and optional 48 hour growth readout (pH color change)
• Indicated for use in 250°F (121°C) gravity sterilization cycles ≥40 minutes and 270°F (132°C) vacuum assisted steam sterilization cycles ≥4 minutes
• Fluorescence detection by 3M™ Attest™ Model 290 Autoreader incubator/reader with a set point of 60 ± 2°C and excitation 340-380 nm / emission 455–465 nm
• Product certification values:
Lot # Population D121°C Z-valueSurvival
TimeKill
Time
2013-01 DF
(Lot A)2.2 x 106
CFU/strip1.8
minutes 10°C 7.82 minutes
18.62 minutes
2013-01 DL
(Lot B)1.9 x 106
CFU/strip1.8
minutes 10°C 7.7 minutes
18.5 minutes
2013-01 DS
(Lot C)1.0 x 106
CFU/strip1.8
minutes 10°C 7.2 minutes
18.0 minutes
II. TERRAGENE Bionova® BT220 Rapid Readout biological indicator for steam sterilization
• Self-contained BI requiring incubation @ 59 ± 1°C with 3 hour fluorescent readout and 24 hour growth readout (pH color change)
• Indicated for use in vacuum assisted and gravity displacement steam sterilization cycles at 121°C–134°C
• Reader incubator capable of reading the fluorescence emission of the product (detection by reader with stimulation [excitation] 340–380 nm / emission 455-465 nm) or in a similar reader
• Product certification values:
Lot # Population D121°C Z-valueSurvival
TimeKill
Time
RVC 09.2012 5.9 x 106
CFU/strip1.6
minutes 14.5°C 7.4 minutes
16.7 minutes
Note: Only one lot of the TERRAGENE Bionova® BT220 product was available for testing at the time of this study.
Reader/Incubator
3M™ Attest™ Model 290 Autoreader incubator/readersNote: The product insert instructions for the TERRAGENE Bionova® BT220 product stated that the Bionova® BIs should be incubated at 59 ± 1°C for a maximum of 3 hours and that the fluorescence detection be performed by a reader with stimulation (excitation)340-380nm / emission 455–465 nm. However, there was no such known reader available from the TERRAGENE Company for their product. As the 3M™ Attest™ Model 290 Autoreader incubator/reader meets the criteria for incubation and fluorescence detection as stated by TERRAGENE, the 3M™ Autoreaders were used to determine the fluorescent readings for the Bionova® BIs evaluated in this study.
Dry Block Incubators
Specially constructed dry block incubators with set point at 59 ± 1°C and fitted with cooling fans to prevent self-contained BI media evaporation allowing for extended incubation of 7 days
Plate Count Media and Incubators
• Sterile tempered soybean casein digest agar with BACTO Agar (DIFCO Tryptic Soy Agar, Lot 9160882)
• Bacteriological incubator calibrated and operating at 59 ± 1°C
• Bacteriological incubator calibrated and operating at 37 ± 1°C
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Testing procedures
BI Exposures @ 121°C
Twenty 3M™ Attest™ 1292 Rapid Readout Biological Indicators from each of 3 lots and 20 TERRAGENE Bionova® BT220 Rapid Readout biological indicators were placed in a metal rack and exposed in a Joslyn Steam Resistometer using a gravity displacement cycle at 121ºC with exposure times ranging from 7 minutes to 13 minutes. Following completion of each respective cycle, the BIs were removed from the sterilizer, allowed to cool for several minutes and then placed in 3M™ Attest™ Model 290 Autoreader incubator/reader wells after crushing the media ampoule. One non-processed BI was incubated with each sample set as a positive control.
After 3 hours of incubation in the Autoreader/incubators, each unit was examined for a ‘positive’ or ‘negative’ fluorescent reading and the results recorded. The BIs were then transferred to dry block incubators fitted with cooling fans to prevent media evaporation during extended incubation. The BIs were examined for growth as indicated by a pH color change at 24, 48 and 168 hours of incubation and the results recorded as either ‘negative’ or ‘positive’ for growth at each incubation time.
BI Exposures @ 132°C
The same procedure as in the 121°C testing was used for the exposures at 132°C with the following exceptions: Ten 3M™ Attest™ 1292 Rapid Readout Biological Indicators from each of 3 lots and 10 TERRAGENE Bionova® BT220 Rapid Readout biological indicators were placed in a metal rack and exposed in an H&W Steam Resistometer using a vacuum assisted cycle at 132ºC with exposure times ranging from 10 seconds to 3 minutes. Note: Only 10 BIs per sample set from each product lot were used for the testing at 132°C due to the limited number of samples available for testing.
Fluorescent False Negative Determination
A BI with a negative 3 hour fluorescent reading that subsequently demonstrated a pH color change indicating growth at 24, 48 or 168 hours was considered to be a ‘fluorescent false negative’ (FFN) response.
Population Count Determination
The spore strips were removed from 10 units of each of 3 lots of 3M™ Attest™ 1292 Rapid Readout Biological Indicators and the 1 lot of TERRAGENE Bionova® BT220 Rapid Readout biological indicators. The spore strips were placed in separate Waring blender jars (with screw cap lids) containing 100 ml of phosphate buffered water. The sample sets were blended for 2 minutes and 10 ml was immediately removed from each blender jar and transferred to separate milk dilution bottles containing 90 ml of phosphate buffered water. Following thorough shaking of the milk dilution bottles, subsequent 1.0 ml transfers were made into test tubes containing 9 ml of phosphate buffered water and homogenized using a Vortex mixer to produce duplicate 10-4 dilution tubes. One 10-4 dilution tube for each sample set was then ‘heat shocked’ by placing it in a water bath set at 100°C for 15 minutes. 10-5 and 10-6 dilutions were then prepared from both the non-heat shocked and heat shocked 10-4 dilution tubes.
Aliquots from each of the 10-4, 10-5 and 10-6 dilutions were transferred into petri dishes in duplicate and 15-20 ml of tempered soybean casein digest agar was poured into each petri dish and thoroughly mixed with the inoculums. Separate sets of petri dishes were then placed in bacteriological incubators with set points at 59 ± 1°C and 37 ± 1°C respectively. The plates were then counted at 48 hours using a colony counter. The colonies from each countable plate were multiplied by the appropriate dilution factor and then averaged within each sample set to determine the population expressed as colony forming units (CFU) per BI for each lot of product. Both non-heat shocked and heat shocked counts were recorded.Note: This method for population count is in compliance with the method for Determination of viable count described in ANNEX A, ISO 11138-1.
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D121 Value Determination
The D121 value determination was based on fraction negative pH color change results at 121°C after 48 hours incubation for all the product lots tested. D121 values were then calculated using the Stumbo- Murphy-Cochran-Procedure as specified in ISO 11138-1 Annex D:9
D Value =
Where:
t = time of exposure
NO = Initial Geobacillus stearothermophilus spore population
Nu = ln (natural log)
t
Log10 NO – Log10 Nu
Number of samples
Total number of negative units
All of the determined D121 values within each separate product lot were averaged to determine the mean D121 value for that lot.
ResultsThe results of the exposures conducted at 121°C are presented in Table 1. Overall, the Bionova® BIs demonstrated a lower resistance to the steam sterilization process at this temperature than the Attest™ BIs. The Bionova® BIs showed predominately negative 3 hour fluorescent readings at all exposure times and high numbers of negative pH color change results in all cycles except the 7 minute exposure time. In contrast, the Attest™ BIs did not show any negative 3 hour fluorescent results until the 12.5 minute exposure and no negative pH color change results until the 11.5 minute exposure time.
Table 1. Results of 3M™ Attest™ 1292 Rapid Readout Biological Indicators and TERRAGENE Bionova® BT220 Rapid Readout biological indicators in side by side exposures at 121°C in a steam resistometer. (N=20)
Number Units Positive/20 Tested @ 121°C
Exposure Time Product
3 Hour Fluorescent
pH Color Change
(Growth)
Fluorescent False
Negative
7 Minutes
1292 Lot A 20 20 0
1292 Lot B 20 20 0
1292 Lot C 20 20 0
Bionova 8/191 19/19 11/19
9 Minutes
1292 Lot A 20 20 0
1292 Lot B 20 20 0
1292 Lot C 20 20 0
Bionova 2 11 9
10 Minutes
1292 Lot A 20 20 0
1292 Lot B 20 20 0
1292 Lot C 20 20 0
Bionova 5 3 4
10.5 Minutes
1292 Lot A 20 20 0
1292 Lot B 20 20 0
1292 Lot C 20 20 0
Bionova 0 7 7
11 Minutes
1292 Lot A 20 20 0
1292 Lot B 20 20 0
1292 Lot C 20 20 0
Bionova 0 2 2
11.5 Minutes
1292 Lot A 20 19 0
1292 Lot B 20 17 0
1292 Lot C 20 19 0
Bionova 0 0 0
12 Minutes
1292 Lot A 20 14 0
1292 Lot B 20 13 0
1292 Lot C 20 2 0
Bionova 0 6 6
12.5 Minutes
1292 Lot A 20 0 0
1292 Lot B 15 1 0
1292 Lot C 10 1 0
Bionova 0 2 2
13 Minutes
1292 Lot A 18 3 0
1292 Lot B 14 1 0
1292 Lot C 9 0 0
Bionova 0 1 1
1Only 19 units in this sample set
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More critically, however, the Bionova® BIs demonstrated a poor correlation between the 3 hour fluorescent readout and the 24–168 hour pH color change (growth) results. The Bionova® product demonstrated a total of 41 fluorescent false negative (FFN) results over the entire exposure range at 121°C with at least one FFN in all but the 11.5 minute exposure. None of the Attest™ product lots showed any FFN results over the entire exposure range. The percentage of FFN results based on the number of pH color change growth positives is contrasted graphically in Figure 1. With the exception of the 11.5 minute exposure time in which no fluorescent or pH color change positives were demonstrated, the Bionova® BI 3 hour fluorescent reading did not detect any subsequent pH color change results in the 10.5–13 minute exposure range. Overall, the Bionova® BI fluorescent reading detected only 11 of the 52 Bionova® BIs that were positive for growth based on the pH color change (~21%).
Figure 1. Comparison of percent Fluorescent False Negative results for the Attest™ and Bionova® BIs with 7–13 minute exposure times at 121°C.
Attest™ 1292 Bionova® BT220
100
90
80
70
60
50
40
30
20
10
0
Exposure Time (Minutes)
Perc
ent F
luor
esce
nt F
alse
Neg
ativ
ess
7 9 10 10.5 11 11.5 12 12.5 13
The results of the testing conducted at 132°C are presented in Table 2. The Bionova® product demonstrated only one FFN result at the 132°C temperature (30 second exposure), but was also shown to have a lower overall resistance at this temperature as well when compared to the Attest™ product (Figure 2). The Attest™ BIs did not show any negative fluorescent results until the 2.5 minute exposure and demonstrated at least partial positive fluorescent results at all exposures. The Bionova® BIs showed one negative fluorescent reading at the 30 second exposure and had zero positive fluorescent readings after the 2.0 minute exposure time. Negative pH color change results for growth were observed at the 1.0 minute exposure for Bionova® BIs while no negative pH color change results were observed for the Attest™ BIs until the 2.0 minute exposure. No FFN results were observed with the Attest™ BIs.
Table 2. Results of 3M™ Attest™ 1292 Rapid Readout Biological Indicators and TERRAGENE Bionova® BT220 Rapid Readout biological indicators in side by side exposures at 132°C in a steam resistometer. (N=10)
Number Units Positive/10 @ 132°C
Exposure Time Product
3 Hour Fluorescent
pH Color Change
(Growth)
Fluorescent False
Negative
10 Seconds
1292 Lot A 10 10 0
1292 Lot B 10 10 0
1292 Lot C 10 10 0
Bionova 10 10 0
20 Seconds
1292 Lot A 10 10 0
1292 Lot B 10 10 0
1292 Lot C 10 10 0
Bionova 10 10 0
30 Seconds
1292 Lot A 10 10 0
1292 Lot B 10 10 0
1292 Lot C 10 10 0
Bionova 9 10 1
1.0 Minute
1292 Lot A 10 10 0
1292 Lot B 10 10 0
1292 Lot C 10 10 0
Bionova 10 7 0
8
Number Units Positive/10 @ 132°C
Exposure Time Product
3 Hour Fluorescent
pH Color Change
(Growth)
Fluorescent False
Negative
1.5 Minutes
1292 Lot A 10 10 0
1292 Lot B 10 10 0
1292 Lot C 10 10 0
Bionova 10 9 0
2.0 Minutes
1292 Lot A 10 10 0
1292 Lot B 10 10 0
1292 Lot C 10 8 0
Bionova 3 0 0
2.5 Minutes
1292 Lot A 6 5 0
1292 Lot B 5 5 0
1292 Lot C 5 4 0
Bionova 0/92 0/9 0/9
3.0 Minutes
1292 Lot A 5 5 0
1292 Lot B 10 7 0
1292 Lot C 7 2 0
Bionova 0 0 0
2Only 9 units in this sample set
With one exception, the 168 hour (7 day) pH color change results indicating growth were the same as the 24 hour pH color change results for both products. One Bionova BI at the 10 minute, 121C exposure demonstrated a negative pH color change at 48 hours but showed a positive result at 168 hours.
The results of the testing for population count and D121 values are summarized in Table 3. All of the products tested met their respective label claims for population and D121 value (with both heat shocked and non-heat shocked population counts) per the requirements for retrospective testing of BIs in ISO 11138-1:2006. Additionally, the population and D121 values for both products met the performance requirements for steam BIs specified in ISO 11138-3. Although the label claims were verified, it was noted that the Bionova® product had a lower D121 value than any of the Attest™ product lots. This is consistent with the lower resistance demonstrated by the Bionova® product at both the 121°C and 132°C temperatures but the lower D121 value by itself does not account for the magnitude of the differences in resistance observed between the two products.
Figure 2. Comparison of percent positive results for the Attest™ and Bionova® BIs with 1–3 minute exposure times at 132°C.
Attest™ Fluorescence Bionova® Fluorescence
Attest™ pH Color Change Bionova® pH Color Change
100
90
80
70
60
50
40
30
20
10
0
Exposure Time (Minutes)
Perc
ent P
ositi
ve
1.0 1.5 2.0 2.5 3.0
Att
est
™ F
luore
scen
ceA
ttest
™ p
H C
olo
r C
han
ge
Bio
nova
® F
luore
scen
ceB
ion
ova
® p
H C
olo
r C
han
ge
As referenced in the materials and methods section testing procedure for population count determination, sets of inoculated agar plates were incubated at 37°C in addition to the standard incubation temperature of 56–60°C (even though Geobacillus stearothermophilus organisms will not grow at 37°C). The 37°C incubation was conducted to provide an indication of the purity of the BI spore populations of the two products. No colonies were observed on any of the culture plates from the Attest™ spore strips incubated at 37°C. However, the Bionova® product demonstrated a population count at 37°C that exceeded the Geobacillus stearothermophilus population count determined at the 59°C incubation temperature. The actual count of the non-Geobacillus stearothermophilus contaminant was 9.6 x 106 CFU/unit. Sub culturing, Gram’s staining and microscopic examination of the contaminating organism indicated it to be a Gram positive, spore forming rod shaped bacterium (likely Genus Bacillus) but no further identification was performed. This finding indicates that the Bionova product DOES NOT contain a homogeneous population of Geobacillus stearothermophilus test organisms.
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Table 3. Comparison of population and D121 values for 3M™ Attest™ 1292 Rapid Readout Biological Indicators and TERRAGENE Bionova® BT220 Rapid Readout biological indicators.
Certification Values
ProductPopulationCFU/Unit
D121 Value(Minutes)
1292 Lot A 2.2 x 106 1.8
1292 Lot B 1.9 x 106 1.8
1292 Lot C 1.0 x 106 1.8
Bionova 5.9 x 106 1.6
Test Values
Product
Non-Heat Shocked
Population (CFU/Unit)
Heat ShockedPopulation (CFU/Unit)
D121 Value Non-H.S.
Population (Minutes)
D121 Value H.S. Population (Minutes)
1292 Lot A 2.2 x 106 3.7 x 106 2.0 1.9
1292 Lot B 1.4 x 106 2.5 x 106 1.8 1.8
1292 Lot C 1.4 x 106 3.4 x 106 1.8 1.7
Bionova 1.2 x 107 8.3 x 106 1.4 1.5
One of the Bionova® non-processed BI controls (sample set for the 10 minute exposure at 121°C) showed a positive growth result after 24 hours of incubation but then turned back to a purple color (negative) after continued incubation. This phenomenon is referred to as ‘reversion’ and can be caused by contaminating organisms that metabolize growth media nutrients which neutralize the acid initially produced by the Geobacillus stearothermophilus organisms and thereby cause an increase in the pH and the reverse color change.
Label claims and efficacy testing for moist heat BIs are generally conducted at 121°C in saturated steam cycles using a resistometer complying with the ISO 18472 resistometer standard. The use of a resistometer not only provides reproducible exposure conditions from cycle to cycle but is also a requirement for testing of BIs claiming compliance to the appropriate ISO 11138 BI standards. Both of the products tested in this study claim compliance with ISO 11138 Parts 1 and 3.9,10
Even though both of the products tested in this study met the performance criteria for population and D121 value per ISO 11138-3, there was a notable difference in the respective resistances of the two BIs as evidenced by the testing results at 121°C and 132°C. The Attest™ BIs clearly showed a greater resistance and challenge at these cycle temperatures than did the Bionova® product. This resistance difference was manifested by both the 3 hour fluorescent results as well as the pH color change (growth) results. The most obvious significance of this difference in resistance is that the Bionova® product would seem to be less likely to detect a shortened exposure time error in a cycle as compared to the Attest™ product. Relative to this issue, it should be noted that the ANSI/AAMI ST79, A1:2010 standard lists ‘insufficient time at temperature’ as one of the reasons for steam sterilization process failures. Causes for this type of failure are indicated as out-of-calibration control timer, inappropriate cycle parameters for the load being processed and oversized load.1
The correlation between the 3 hour fluorescent reading and a later pH color change result is critical relative to the use of any rapid readout BI. A negative 3 hour fluorescent reading with a subsequent positive reading for growth indicated by a pH color change with additional incubation time, i.e., an FFN, represents the ‘worst case’ scenario for use of a rapid readout BI. The consequence of releasing a product load for use in a health care facility based on a FFN result could create a potential for distribution of inadequately processed items thereby creating a patient safety risk. Of the 179 Bionova® BIs tested at 121°C, 41 BIs demonstrated FFN results (~23%). If only the total number of Bionova®
Discussion
1 0
BIs that were positive for growth (52) is considered, the percentage of FFN for the Bionova® product increases to ~79%.
The current guidelines for reduced incubation of a BI, i.e., an incubation time of less than 7 days, are specified in a United States (U.S.) Food and Drug Administration (FDA) document for validation of BI incubation time.11 The U.S. FDA criterion for a BI incubation time of less than 7 days is that the reduced incubation time must demonstrate a correlation of greater than 97% compared to growth after 7 days of incubation. (The ‘sensitivity’ calculation used to demonstrate this correlation is stated as such in the Bionova® BT220 product insert). Based on the Bionova® FFN and pH color change (growth) positives from the 121°C testing, the 3 hour readout had a 22.6% correlation with the 7 day pH color change results. This 22.6% correlation value is significantly less than the ≥ 97% criterion specified by the U.S. FDA and is contrary to the ≥ 97% sensitivity statement in the Bionova® product insert. By comparison the 3 hour fluorescent reading of the Attest™ BIs detected 100% of the BIs that were positive for pH color change indicating growth.
The use directions in the product insert for the Bionova® BIs state that: “the final readout of a negative result should be made after (3) three hours of the indicator incubation”. Although a caution is made to incubate the BI for 24 hours, it is questionable that many users would continue incubation after the 3 hour reading. The value in using a rapid readout BI is to minimize the time necessary for sterility release of processed items and ultimately shorten the overall instrument turnaround time. If processed items were to be held for 24 hours, it seems unlikely that a facility would choose to pay a higher cost for a rapid readout BI when a conventional BI would serve the same purpose.
The presence of a high population of a non-test organism (or organisms) was an unexpected finding in this study. ISO 11138-1 clearly states in section 5.3.2 “Only one strain of test organism shall be used in a batch of inoculated carriers, unless the manufacturer has demonstrated that the use of multiple strains does not significantly affect test organism performance in the specified sterilization process.” While it is unknown what documentation the manufacturer has in place relative to this issue, there is a legitimate concern as to possible interference with the Geobacillus stearothermophilus readouts by the non-test organism and ultimately its impact on the reliability of this product.
An additional consequence relative to the presence of an organism (or organisms) that grows at 37°C is the potential for a positive growth result in a non-processed BI control that is incubated in a defective incubator. The Bionova® BT220 product instructions clearly state that it is important to “use a non-sterilized biological indicator as a positive control every time a processed indicator is incubated. The positive control ensures that correct incubation conditions were met.” Geobacillus stearothermophilus ATCC 7953 organisms as used in the Bionova® BI will not grow below 45°C whereas the non-test organism present in the Bionova product will grow at incubation temperatures below 45°C. An incubator operating at less than the 56-60°C set point, i.e., below 45°C, might well allow for growth in the non-processed BI control indicating the incubator was working properly. In this situation, Geobacillus stearothermophilus organisms that survived as a result of inadequate processing would not grow and the user would have no indication of a possible sterilization process failure.
11
Two rapid readout biological indicators for monitoring the steam sterilization process, the 3M™ Attest™ 1292 Rapid Readout Biological Indicator and the TERRAGENE Bionova® BT220 Rapid Readout biological indicator for steam sterilization were evaluated side by side in steam resistometers using 121°C and 132°C cycles. The Attest™ BIs demonstrated greater resistance to the steam sterilization process in both the 121°C and 132°C cycles than the Bionova® by requiring long sterilization exposure times in order to be killed. A BI that has a higher resistance will provide a greater ability to adequately monitor the cycle.
The Bionova® BIs showed a high number of fluorescent false negative (FFN) results at 3 hours in the 121°C cycles and one FFN at 132°C compared to zero FFN for the Attest™ BIs at both 121°C and 132°C. As release of a product load based on a FFN result could lead to distribution of inadequately processed items within a health care facility, this is considered to be a patient safety issue. The presence of a high population of a non-test organism (or organisms) on the Bionova® BI spore strip is both a concern for possible interference of the Geobacillus stearothermophilus readouts and for potential erroneous readings of non-processed BI controls.
Although the two products evaluated in this study are described and presented to the marketplace as being identical, they are not equivalent products in terms of performance for the monitoring of steam sterilization cycles. Based on the outcomes of the testing in this study, most notably the high incidence of FFN results and the presence of large numbers of non-test organisms, it could be concluded that use of the TERRAGENE Bionova® BI presents a potential liability relative to the release of steam sterilized loads in health care facilities.
Summary Conclusions
Please recycle. Printed in U.S.A. Bionova and Terragene are trademarks of Terragene, SRL.3M and Attest are trademarks of 3M.© 3M 2011. All rights reserved.70-2010-8353-5
Infection Prevention Division3M Health Care3M Center, Building 275-4E-01St. Paul, MN 55144-1000U.S.A.1 800 228-3957www.3M.com/infectionprevention
1. Association for the Advancement of Medical Instrumentation. ANSI/AAMI ST79, Comprehensive guide to steam sterilization and sterility assurance in health care facilities. An amendment to the standard, A1:2010. AAMI, Arlington, VA.
2. Association of periOperative Registered Nurses: Perioperative Standards and Recommended Practices. AORN, Denver, CO, 2010.
3. Centers for Disease Control and Prevention. Guideline for Disinfection and Sterilization in Health Care Facilities. CDC, Atlanta, GA, 2008.
4. Maki DG, Hassemer CA. Flash sterilization: carefully measured haste. Infection Control 1987;8: 307-310.
5. Rutala WA, Gergen MF, Weber DJ. Evaluation of a rapid readout biological indicator for flash sterilization with three biological indicators and three chemical indicators. Infection Control Hospital Epidemiology 1993;14:390-394.
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8. International Organization for Standardization (ISO). Medical Devices — Sterilization of health care products — Biological and chemical indicators — Test equipment: Geneva, Switzerland: ISO 2005. ISO 18472.
9. International Organization for Standardization (ISO). Medical Devices — Sterilization of health care products — Biological indicators — Part 1: General Requirements, 2nd ed. Geneva, Switzerland: ISO 2006. ISO 11138-1.
10. International Organization for Standardization (ISO). Medical Devices — Sterilization of health care products — Biological indicators — Part 3: Indicators for moist heat sterilization, 2nd ed. Geneva, Switzerland: ISO 2006. ISO 11138-3.
11. Center for Devices and Radiological Health, United States Food and Drug Administration, Guidance for Biological Indicator (BI) Premarket Notification [510(k)] Submissions, Attachment II — Recommended Validation of Biological Indicator Incubation Time, Washington, DC: CDRH 2007.
Note: This study was funded by 3M Health Care
Philip SchneiderPhilip Schneider is a microbiologist with over 40 years of professional experience in sterilization and disinfection applications in health care. He is currently working as a senior consultant with LexaMed, Toledo, OH, a company involved with sterilization, microbiology and regulatory support for the medical device and pharmaceutical industries. He is the Convener of the ISO/TC 198 Biological Indicator Working Group 4. Additionally he is the Co-chair of both the ANSI/AAMI Biological Indicator and the Ethylene Oxide Hospital Practices Working Groups. In these standards organization roles he has directed activity for development of all of the currently published ISO and ANSI/AAMI standards for the manufacture and use of biological indicators.
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