When bacteria are placed under stress they can create a protective coating and turn into spores, for example Bacillus spp and Clostridium spp – in effect they go into hibernation. Spores can lie dormant for long periods of time before germinating to become vegetative bacteria.
The main problem with spores is their resistance to many of the standard disinfectants that are used. Traditional disinfectants, such as alcohols, quaternary ammonium compounds, phenols and amphoteric surfactants, are effective against bacteria in their vegetative state but do not kill spores. Even some experienced pharmacists do not realise that alcohol does not kill spores.
There are two main issues: maintaining a spore-free environment and preventing spore contamination from the use of disinfectants.
Spores can enter the cleanroom on people and on components at a surprisingly high rate. Annex 1 of Good Manufacturing Practice (GMP) confirms that “The transfer of materials into and out of the unit is one of the greatest potential sources of contamination”.(1)
Research has shown that around 40% of consumables passed into critical areas (eg, sharps bins) are contaminated with bacterial spores.(2) The same study also identified that, if the transfer disinfection procedure employed was spraying with alcohol, only 27.6% of the spores would be removed.
Steps to maintain a spore-free environment
Reduce initial bioburden
There are two ways in which the initial bioburden (and therefore the risk of spores) can be reduced:
- Using a spray-and-wipe protocol for transfer disinfection has been shown to improve significantly the effectiveness of the transfer procedure.(2) However, this can be time- consuming if there are lots of individual items, such as syringes.
- Paper packaging should be eliminated where possible because, in addition to creating unwanted particulates, it harbours contamination and is difficult and time-consuming to spray and wipe effectively.
Multipacks of consumables in polythene packaging are an excellent way of achieving both these aims. As well as being paper-free, they significantly speed up the transfer process as they are quick and easy to spray and wipe. They also reduce the amount of alcohol and wipes that need to be used for spraying and wiping. Consider multipacks for any bulk items or paper-packaged items, such as needles, syringes, pens, waste bags, bins, medical devices, chemotherapy spill mats and environmental testing swabs.
Remove spore contamination from the critical area
Disinfectants that have sporicidal activity (sporicides) include aldehydes, hypochlorites and hydrogen peroxide/peracetic acid blends. These are often toxic, irritant, corrosive, leave residues or need a long contact time. Many sporicides are oxidising agents, and the environmental impact of using these in a cleanroom containing expensive equipment has to be carefully considered. When validating disinfectants, a review should be undertaken of the available data for corrosion testing on materials used in cleanrooms, isolators and laminar flow cabinets.
However, a unique sporicide is available that offers a safe and effective solution. It is an alcohol-free blend of a quaternary ammonium compound and stabilised chlorine dioxide. It is nontoxic and noncorrosive, with low odour and a required contact time of only five minutes.
While efficacy is important, it is also crucial to consider the final specification of the sporicide. The user must ensure it can be transferred easily into the cleanroom and that it meets the required standards for sterility. Current GMP states that “disinfectants in Grade A and B cleanrooms should be sterile prior to use”.(1)
Serious consideration should be given to the inclusion of a sporicide as part of the regular disinfection regime.
Preventing spore contamination from disinfectants
Certain presentations of disinfectants could contaminate your critical area. How can this happen?
The first thing is to ensure that the disinfectant you choose is guaranteed spore-free, and disinfectants for Grade A and B areas should be sterile. Ensure that you obtain full supporting data from the manufacturer, validating their production method and the sterility of the product.
The second issue is the in-use shelf-life of the disinfectant. In most cases, disinfectants are delivered by either trigger sprays or aerosols. Trigger spray systems offer significant advantages over aerosols and these include:
- An adjustable nozzle so liquid can be dispensed as a jet or a spray.
- An environmentally-friendly format, as they contain no propellants.
- An economical solution, as all of the liquid can be dispensed from a trigger spray, so there is no wastage.
However, there is a significant potential drawback with some trigger sprays, as contaminated air can be drawn back into the bottle, compromising the sterility of the liquid.
Validation work in the licensed pharmacy unit of the Queen Elizabeth Hospital in Birmingham identified that a trigger spray alcohol, used to spray items into an isolator, had been contaminated with fungal spores only eight hours after opening.(3)
A closed trigger spray system can solve this problem, providing the benefits of a trigger spray while guaranteeing the integrity of the contents.
If designed correctly, it will prevent contaminated air from being drawn back into the liquid and so eliminate the chance of contaminating the liquid. However, not all closed trigger spray systems are the same.
Check that the system is validated
An inspector will expect confirmation of the in-use shelf-life of a sterile liquid dispensed in a trigger spray format. It is therefore important to examine the validation work carried out and ask the following questions of the manufacturer:
- Was the trigger spray tested with nutrient broth as well as the sterile fluid?
- Was the testing carried out using a sufficient number of samples?
- Was the test work carried out in a controlled Grade D environment?
- Did negative controls confirm the original samples were not contaminated?
- Was the broth viable to the end of the testing?
- Was the sampling carried out in the way the bottle will be used – through the nozzle?
- Was the sterility testing carried out by an independent laboratory?
So, to control spore contamination:
- Use a spray-and-wipe protocol for all items to be transferred into the critical area.
- Consider the use of polythene packaging instead of paper.
- Ensure disinfectants are sterile and spore-free.
- Prevent spore contamination from the disinfectant by using a fully validated trigger spray system.
- Include a dedicated sporicide in your regime.
If all these areas are acted upon then it should be possible to maintain a spore-free environment in the cleanroom.
- Eudralex. Vol 4 Good Manufacturing Practice, Annex 1, Manufacture of Sterile Medical Products Rev; 2003.
- Cockcroft MG, Hepworth D, Rhodes JC, et al. Validation of liquid transfer disinfection techniques for transfer of components into hospital pharmacy cleanrooms. Hosp Pharmacist 2001;8:226-32.
- Contamination control at your fingertips. Pharmaceutical Manufacturing Int Autumn 1999.