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 Table of Contents  
MINI REVIEW
Year : 2019  |  Volume : 21  |  Issue : 2  |  Page : 66-69

Advantages and limitations of rapid biological indicator for fast sterilisation assurance


1 Department of Central Sterile Supply, Tata Medical Center, Kolkata, West Bengal, India
2 Department of Microbiology, Tata Medical Center, Kolkata, West Bengal, India

Date of Submission05-Jul-2018
Date of Decision30-Oct-2019
Date of Acceptance04-Nov-2019
Date of Web Publication17-Jan-2020

Correspondence Address:
Mr. Debabrata Basu
Department of Central Sterile Supply, Tata Medical Center, 14, Major Arterial Road (E-W), New Town, Kolkata - 700 160, West Bengal
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jacm.jacm_20_18

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  Abstract 


Continuous quality monitoring in sterilisation processes is paramount importance for supplying sterile materials to the patients. The sterilisation monitoring is required because early prediction of steriliser malfunctioning is impossible, and malfunction gets detected only when it runs. Internationally there are three types of sterilisation monitoring system such as physical, chemical and biological monitoring. The physical monitoring system is dependent on time, temperature and pressure. The chemical and biological monitoring systems are also dependent on the same physical parameters but in addition require the presence of condensing steam for sterility assurance. The main aim of this article is to elaborate differences between the 'conventional biological indicator' and the 'rapid biological indicator' with their benefits and disadvantages for biologically proven sterility assurances. However, it is not possible to determine whether biological material in the indicator is alive or dead, visually. The article explains that the detection of living spores can be technically challenging, and each and every step should be monitored carefully because spores are highly resistant to harsh environmental conditions. The results show that proper incubation time in an appropriate growth medium and optimal temperature is the only way to detect the living microorganisms in a biological indicator for proper sterility assurance.

Keywords: Biological indicator, chemical indicator, physical parameter, rapid auto-reader, sterilisation monitoring


How to cite this article:
Basu D, Bag SC, Mukherjee S, Goel G. Advantages and limitations of rapid biological indicator for fast sterilisation assurance. J Acad Clin Microbiol 2019;21:66-9

How to cite this URL:
Basu D, Bag SC, Mukherjee S, Goel G. Advantages and limitations of rapid biological indicator for fast sterilisation assurance. J Acad Clin Microbiol [serial online] 2019 [cited 2020 May 31];21:66-9. Available from: http://www.jacmjournal.org/text.asp?2019/21/2/66/276119




  Introduction Top


Continuous quality monitoring of sterilisation processes is of paramount importance to supply sterile materials for patient care purposes. The monitoring is very much required because detection of steriliser malfunctioning is technically challenging when the steriliser is not in operation and detection of malfunction or appropriate operation occurs only when the steriliser runs. There are three types of monitoring systems i.e., physical, chemical and biological monitoring. The physical monitoring system consists of time, temperature and pressure. The chemical and biological monitoring system is also dependent on the same physical parameters but in addition includes the action of condensing steam for sterility assurance. The objective of this article is to differentiate the conventional 'Biological Indicator' and the rapid 'Biological Indicator' with their benefits, disadvantages and possible alternatives that helps to evaluate sterilisation processes for optimal sterility assurance.

The biological monitoring system depends on live non-pathogenic bacterial spores with stringent sterilisation parameters. Biological indicators (BIs) are defined in the European and International Standards (ENISO 11138 series). The indicators are prepared using bacterial spore strip containing 106 living spores. It is not possible to determine whether BIs are alive or dead visually. The resistance characteristics of each individual germ are defined by the decimal reduction value (D-value), which is the time needed to reduce the population of a BI to one-tenth of the original population.[1] During the sterilisation process, the spore population always decreases due to the exponential kill characteristic of a steriliser. However, the population will never reach an absolute zero value. Therefore, modern definitions of goods declared 'sterile' do not indicate the absolute absence of biological activity but determine aseptic conditions with a certain probability, called sterility assurance level, which is <10−6 according to EN 554.


  Resistance of Bacterial Spore Top


Vegetative bacteria are the bacterial cells that can metabolise and undergo binary fission when nutrients are available. Some bacterial classes (Bacillus and Clostridia) are able to form so-called endospores that are highly resistant to harsh environmental conditions. The resistance characteristics of bacterial spores depend on various factors. Some of these factors may be intrinsic to the microorganism itself (such as nutrient deficiency, pH factor, cell thickness and temperature), sporulation system (e.g., solid-state or submerged fermentation) or different carriers (used in BI) such as plastic, paper, metal or high-density polyethylene fibres (i.e., Tyvek). Basically, endospores are 'resting bacteria' but can be activated to become vegetative cells if favourable conditions are available.[2]

There are two main types of BIs:

Conventional biological indicator

BIs are self-contained and consist of a filter paper, a dry spore strip, a porous material, growth media and a pH indicator. For conventional BI, the detection of living spores is easy because during incubation vegetative bacteria metabolise using the growth media (a chemical reaction between vegetative cell enzyme with external growth media i.e., Tryptic Soy Broth that is used in BI) to form acids that change media colour by changing the pH value. This change shows that the spores are alive.[3] Although in some instances with an exposure near the resistance of a BI, it is occasionally observed that some spores may be alive (sub-lethal damage of spores due to poor steriliser condition) and multiply after a longer time of incubation (>8–12 h). Hence, ideal recommendations for safe practices are at least 72 h to 168 h of incubation (three to seven days). These types of damaged spores may 'repair' themselves to grow into vegetative cells and start metabolism later on. Under those conditions, vegetative cell enzyme production is possible when spores return to vegetative state.[4],[5]

Rapid biological indicator

The rapid BI has a dual readout system. The conventional system and the rapid readout system. The rapid system detects the presence of active alpha-glucosidase enzyme (alpha-glucosidase from Bacillus stearothermophilus, ATCC 7953 which is partially integrated into the outer matrix of the spore cortex where most spore associated enzymes [e.g., amylases] are involved in activation, germination and outgrowth. Their function being the preparation of nutrition for the bacterial cell) in the spore cortex, which suggests that it is necessary in the early stage of germination and outgrowth prior to new enzyme synthesis. The maximum period of activity of alpha-glucosidase enzyme is 1–3 h after the steam sterilisation process, only if the spores are alive.[6],[7] For rapid sensing in rapid BI a chemical (4-methylumbelliferone or 4-MU) is added with alpha-glucosidase enzyme which produces fluorescence after being split by the enzyme to 4-methylumbelliferyl-alpha-D-glucoside. The enzyme splits (enzymes are not very stable over time if exposed to humid environmental conditions) the alpha-glucosidase bond and sets 4MU free for detection. This fluorescence can be detected by the rapid auto-reader (a special fluorescence sensing incubator) within a very short period (pass-fail result of rapid BI is less than three hours) [Table 1].
Table 1: Compare between conventional and rapid biological indicator

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  Limitations of the Rapid Biological Indicators System Top


If the spores suffer sub-lethal damage due to the heat, there is a rare possibility that alpha-glucosidase enzyme may or may not be produced or may even be destroyed (soluble alpha-glucosidase outside of spores cortex is rapidly removed by heat activation).[8] However, even if the spores are severely damaged by heat or other reasons, they can 'repair' themselves because they are highly resistant to any adverse environmental condition. When favourable conditions (such as appropriate incubation temperature or contact with external growth media) are achieved then there is a high possibility of damaged spores getting activated to a vegetative cell again.

Therefore, the short period available to detect fluorescence is not adequate for sub-lethally damaged spores to 'repair and convert' them to vegetative cells. Hence, the rapid BI has the potential to incorrectly predict adequate sterilisation and incorrectly supply unsterile loads.[9],[10],[11]

A test was performed in GKE laboratory in Germany using 100 numbers of BIs to check the delayed growth of vegetative cell enzyme. The organism used in this study was Geobacillus stearothermophilus (ATCC 7953). A BI Evaluation Resistometer (vessel) was used to maintain the accurate sterilisation time of growth and survival-kill factor of BIs. The sterilisation process was carried out at 121°C for 15 min with 2 bar pressure. After the sterilisation process, the BIs were kept in a conventional incubator. It was observed that 4/100 (4%) BIs showed positive result after 72 h of incubation (positivity was flagged between 120 to168 h).[12],[13],[14]


  Conclusion Top


A BI is denoted as a 'load control indicator' (used in every load) that verifies the entire load by indicating the killing of microorganisms by the sterilisation process and gives assurance that the load is sterile (FBIO-value). The microorganisms used in the BI are all non-pathogenic bacterial spores which are most resistant in a specific sterilisation process. BIs are the only process indicators that can directly monitor the lethality of a given sterilisation process when all critical variables in the steriliser parameters (e.g., time, temperature and chemical concentration of sterilising agent) are unidentified.[15] We conclude that rapid BIs should not be used as a substitute for the conventional BI. Materials may be issued after confirmation from validated type-V Chemical Indicator (ISO 11140-1) result which is equivalent to, or exceed the performance requirements (as per resistometer) given in the ISO 11138 series for BI.[15],[16]

Acknowledgement

The test mentioned in this study was done in the GKE laboratory (GKE - GmbH, Auf der Lind 10, D-655529 Waldems, Germany). We are grateful for this technical support.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Basu D, Bhattacharya S, Mahajan A, Ramanan VR, Chandy M. Sterilization indicators in central sterile supply department: Quality assurance and cost implications. Infect Control Hosp Epidemiol 2015;36:484-6.  Back to cited text no. 1
    
2.
Biological Indicators: Measuring sterilization. Available from: http://202.74.245.22:8080/xmlui/bitstream/handle/123456789/1014/Chapter%2013-Biological-indicators- Measuring-sterilization.pdf?sequence=15. [Last accessed on 2018 May 09].  Back to cited text no. 2
    
3.
Neumann O, Feronti C, Neumann AD, Dong A, Schell K, Lu B, et al. Compact solar autoclave based on steam generation using broadband light-harvesting nanoparticles. Proc Natl Acad Sci U S A 2013;110:11677-81.  Back to cited text no. 3
    
4.
Live-Imaging of Bacillus Subtilis Spore Germination and Outgrowth. Available from: https://pure.uva.nl/ws/files/2451269/151494_04.pdf. [Last accessed on 2018 May 09].  Back to cited text no. 4
    
5.
Setlow B, Korza G, Blatt KM, Fey JP, Setlow P. Mechanism of bacillus subtilis spore inactivation by and resistance to supercritical CO2 plus peracetic acid. J Appl Microbiol 2016;120:57-69.  Back to cited text no. 5
    
6.
Vesley D, Nellis MA, Allwood PB. Evaluation of a rapid readout biological indicator for 121 degrees C gravity and 132 degrees C vacuum-assisted steam sterilization cycles. Infect Control Hosp Epidemiol 1995;16:281-6.  Back to cited text no. 6
    
7.
Albert H, Davies DJ, Woodson LP, Soper CJ. Biological indicators for steam sterilization: Characterization of a rapid biological indicator utilizing Bacillus stearothermophilus spore-associated alpha-glucosidase enzyme. J Appl Microbiol 1998;85:865-74.  Back to cited text no. 7
    
8.
Huesca-Espitia LC, Suvira M, Rosenbeck K, Korza G, Setlow B, Li W, et al. Effects of steam autoclave treatment on Geobacillus stearothermophilus spores. J Appl Microbiol 2016;121:1300-11.  Back to cited text no. 8
    
9.
Lukomskaya IS, Voznyi YV, Lanskaya IM, Podkidisheva EI. Use of beta-maltosides (p-nitrophenyl-beta-D-maltoside, 2-chloro-4-nitrophenyl-beta-D-maltoside and 4-methylumbelliferyl-beta-D-maltoside) as substrates for the assay of neutral alpha-glucosidase from human kidney and urine. Clin Chim Acta 1996;244:145-54.  Back to cited text no. 9
    
10.
Matsui T, Shimada M, Saito N, Matsumoto K. Alpha-glucosidase inhibition assay in an enzyme-immobilized amino-microplate. Anal Sci 2009;25:559-62.  Back to cited text no. 10
    
11.
Setlow B, Korza G, Setlow P. Analysis of α-glucosidase enzyme activity used in a rapid test for steam sterilization assurance. J Appl Microbiol 2016;120:1326-35.  Back to cited text no. 11
    
12.
Understanding Biological Indicator Grow-Out Times-Part II. Available from: http://www.pharmtech.com/understanding-biological-indicator-grow-out-times-part-ii [Last accessed on 2019 Jun 10].  Back to cited text no. 12
    
13.
Alfa MJ, Olson N, DeGagne P, Jackson M. Evaluation of rapid readout biological indicators for 132 degrees C gravity and 132 degrees C vacuum-assisted steam sterilization cycles using a new automated fluorescent reader. Infect Control Hosp Epidemiol 2002;23:388-92.  Back to cited text no. 13
    
14.
Design, Validation and Monitoring of Sterilization Processes and Instrument. Available from: https://www.ciet-bio.com/?forcedownload=1&no_encrypt=1&file=filesystem%2FInstant++BI++CI+Class+5+%D7%95%D7%95%D7%90%D7%9C%D7%99%D7%93%D7%A6%D7%99%D7%94+.pdf. [Last accessed on 2019 Oct 08].  Back to cited text no. 14
    
15.
Guideline for Disinfection and Sterilization in Healthcare Facilities; 2008. Available from: https://www.cdc.gov/infectioncontrol/guidelines/disinfection/sterilization/sterilizing-practices.html. [Last accessed on 2019 Apr 09].  Back to cited text no. 15
    
16.
Ling ML, Ching P, Widitaputra A, Stewart A, Sirijindadirat N, Thu LT, et al. APSIC guidelines for disinfection and sterilization of instruments in health care facilities. Antimicrob Resist Infect Control 2018;7:25.  Back to cited text no. 16
    



 
 
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