By Sarah Joanna Hiom, PhD, MRPharmS
To compare a new total immersion method for validating aseptic transfer with a conventional roll plate method, and to use the new method to determine an optimum disinfection technique for use in hospital aseptic dispensing units.
An Envair Micro-ISO isolator (set at 0.39 m/s positive airflow velocity) was used in a Grade D environment.
Assessment of bioburden on plastic ampoules, before and after aseptic transfer into an Envair isolator.
Enumeration of colony forming units per ampoule before and after disinfection by four different techniques.
The new, total immersion method was approximately 6 times more sensitive than the roll plate method. That sensitivity allowed four different disinfection techniques to be compared. Significant differences were seen and wiping was observed to be a crucial step.
The described method can be used to assess different aseptic disinfection transfer techniques. Of those tested, statistical analysis showed that wiping with isopropyl alcohol alone or in conjunction with 70 per cent industrial methylated spirit (IMS) spraying showed significant advantage over spraying alone and that spraying with IMS was as effective after 10s exposure as after 2 min. This total immersion enumeration method may also be used for training and in-house validation of staff techniques.
Hospital aseptic dispensing units must provide products that are sterile and have assured quality. Owing to the nature of aseptic dispensing activity, limited or no final aseptic testing is undertaken before product release. Therefore, all assurance of quality must be provided by adequately validated systems of work. Current guidelines concerning environmental monitoring of these units include acceptable limits for the presence of viable cells.1,2
These guidelines are not comprehensive and the NHS Quality Control Committee has reported: “Where definitive standards do not exist, particular importance should be attached to obtaining meaningful results, monitoring trends and setting in-house standards.”2
Clean areas for the manufacture of aseptic products are classified according to the required characteristics of the environment. Classifications range from Grade A for high-risk operations in the controlled work zone down to Grade D for less critical pre-preparation stages.1
Commonly used hospital aseptic disinfection transfer systems consist of a combination of spraying with sterile 70 per cent alcohol and wiping with sterile disinfectant soaked cloths, eg, isopropyl alcohol (IPA) wipes.
Some hospital isolators are also programmed so that the transfer hatch is “locked” for a set period. This has the dual purpose of allowing adequate “clean air” changes within the hatch before the inner doors are opened and also allows adequate contact time between the item and disinfectant before manipulations are carried out. The period for effective exposure to 70 per cent alcohol spray has often been queried. Therefore, included in the disinfection programmes are different exposure times to 70 per cent industrial methylated spirit (IMS).
In-house validation of these systems usually consists of either the roll plate method or swabbing before and after disinfection. These are relatively insensitive techniques used as part of the total environmental monitoring process in an attempt to detect any gross patterns of change in bioburden.
If numbers of microbes could be counted before and after manipulation this would not only allow an accurate means of validating disinfection programmes but could also be used to monitor various aseptic parameters, eg, bioburden of supplied items, effect of various in-house storage conditions and staff techniques and training.
The transfer of small components used in the hospital aseptic manufacturing process from preparation areas to the controlled work zone incorporates a disinfection process. The aim of this project was to determine and employ an accurate method to validate these transfer disinfection programmes. A method to quantify bioburden accurately was investigated and compared with a conventional roll plate technique.
Total immersion enumeration
A single ampoule (5ml Steri-Amp of water for injections BP, Steripak Ltd, Runcorn, Cheshire) was immersed in 30ml water for injections (WFI) contained in a sterile 50ml centrifuge tube. The ampoule was then vortexed and sonicated in turn for one minute, washed with 2ml WFI and removed. The water remaining in the tube was pour-plated 1:1 with molten double strength nutrient agar (Oxoid, Basingstoke, Hampshire), incubated at 30C for seven days then enumerated for colony forming units (CFUs).
Roll plate enumeration
Predetermined surface areas of the ampoule (50 per cent of the body) were exposed to contact plates (Biotest Hycon TC, Germany) for five seconds each. Plates were enumerated for CFUs after incubation at 30C for three days.
Fifty ampoules were exposed to natural environmental contamination in a busy corridor for two days. Using sterile gloves, batches of ampoules (n=5) were then treated with one of four different disinfection programmes, while undergoing aseptic transfer into an Envair Micro-ISO isolator (+ve, 100Pa).
The four disinfection programmes were:
- 1.Ampoules sprayed with 70 per cent IMS (Klercide 30/70, Shield Medicare, Farnham, Surrey) with a two-minute “lock out”
- 2.Ampoules sprayed with 70 per cent IMS, no “lock out”
- 3.Ampoules wiped with 70 per cent IPA (Alcowipes, Seton Healthcare Group Plc, Oldham, Lancashire)
- 4.Ampoules sprayed with 70 per cent IMS and wiped with 70 per cent IPA
The isolator was pre-cleaned with Proceine 40 (AGMA, Haltwhistle, Northumberland) and set at 0.39m/s positive airflow velocity. Surface contamination remaining on an ampoule after each disinfection programme was determined by both the roll plate method and the new total immersion method. Bioburden on ampoules taken straight from the manufacturer’s original packaging was also determined by the new total immersion method.
Environmental monitoring was performed using settle plates and swabs and the environmental integrity was maintained throughout the experiment.
The results showed that, under the conditions stated, the total immersion enumeration technique was approximately six times more sensitive at detecting surface contamination than the roll plate method (Table 1). This sensitivity allowed a more accurate assessment of different disinfection techniques. Analysis of variance on the data (p=0.05) showed that there was a significant difference between the disinfection methods used and the control (natural exposure). Under these experimental conditions, spraying with IMS and no “lock out” actually meant exposure to IMS for approximately 10 seconds. Statistically this showed no difference to spraying with IMS with a two-minute “lock out” period. Spraying with IMS combined with wiping with IPA showed no significant advantage over wiping alone, although both of these were significantly better than spraying alone. Analysis of the results indicated that all disinfection techniques showed at least a 10-fold reduction in microbial bioburden when compared with the natural exposure control.
The bioburden of similar ampoules (n=5) taken straight from their original packaging was determined by the total immersion method and was found to be relatively low at 3.2 (+/-2.7) CFUs/ampoule.
Table 1: Enumeration of colony forming units (CFUs) per ampoule (n=5) before and after various disinfection programmes
|Disinfection method||Enumeration method|
|Roll plate CFU/ampoule (+/-sd)||Total immersion CFU/ampoule (+/-sd)|
|Control (natural exposure)||30.4 (+/- 8.2)||168.6 (+/- 60.2)|
|Spray 70% IMS plus 2 min lockout||0.8 (+/- 1.1)||6 (+/- 2.9)|
|Spray 70% IMS, no lockout||0||4 (+/- 2.8)|
|Wipe with 70% IPA||0||1.2 (+/- 1.3)|
|Spray with 70% IMS plus wipe with IPA||0||0.6 (+/- 0.5)|
The results demonstrate that the described method can be used to enumerate accurately surface bioburden on small manufacturing items and therefore may be used to assess different disinfection transfer techniques.
However, it was necessary in this experiment to compare results of microbes grown on different media and for different incubation times. Microbes determined by the roll plate technique were inoculated on the surface of Biotest TC plates and incubated for three days before counting. Microbes determined by the total immersion technique were embedded in nutrient agar and incubated for seven days before counting. The use of varied incubation times can be rationalised by embedded bacteria taking longer to form visible colonies compared with surface growing bacteria. The use of different media for the two techniques may have produced slightly altered results; however, the media contents are fairly similar and therefore are not considered to be a major confounding factor.
The total immersion method described was intended as a practical tool to validate the disinfection process and therefore did not attempt to recover 100 per cent of microbes present. However, the use of peptone water instead of WFI for washing cells and the use of two different media and incubation temperatures (to allow bacteria and fungi to grow) may provide a more accurate determination of total bioburden, if this is required.
This experiment has demonstrated that ampoules taken straight from their original packaging had a low relative bioburden (3.2 CFUs/ampoule) and that all four disinfection regimens reduced bioburden 10-fold. We could therefore infer from these results that ampoules removed from their original packaging and immediately disinfected would be left with a bioburden of less than 1 CFU/ampoule. Our results also showed that ampoules stored loose in a busy corridor became heavily contaminated. However, outer packaging often consists of cardboard which is known to harbour micro-organisms,3,4
particularly spores. This experiment has also demonstrated that ampoules taken straight from their original packaging had a low relative bioburden and that those stored in a busy corridor became heavily contaminated. The question of when to remove outer packaging has frequently been raised. Further work is therefore required to investigate the effect of cardboard presence, particularly the wax- or lacquer-coated type, on the environment. The described technique, however, may be used to examine the surface contamination from various in-house storage conditions.
This experiment demonstrated that wiping with IPA is as effective as spraying with IMS and wiping with IPA and both are significantly better than spraying alone at reducing bioburden (p=0.05). From this, it can be inferred that wiping is a crucial step in the disinfection process.
Hospital units using the roll plate method in-house to validate their disinfection processes should bear in mind that they are possibly only detecting one sixth of the microbes present; however, contact transfer may be more representative of the contamination risk.
The method described could be used routinely to monitor bioburden and set in-house standards within hospital aseptic dispensing units. This work has indicated that, under the conditions described, spraying and wiping together have no significant advantage over wiping alone, although they are significantly better than spraying alone. We can therefore infer that the most effective and time-efficient aseptic disinfection method tested was to wipe items with IPA-soaked cloths. However, because of the practical difficulty of wiping some items, it is recommended to spray into the controlled work zone with 70 per cent IMS (maintaining the safety margin of a two-minute “lock out”) and subsequently wipe critical areas with IPA prior to manipulations.
The “cleanliness” of supplied and stored items can also be established using the described method and an informed decision then made as to when the cardboard packaging should be removed or how items should be stored. Preferably, ampoules should be removed from their outer packaging as late in the preparation process as possible, ideally without taking cardboard into preparation areas of Grade D or higher and particularly not into the controlled work zone.
The method may also be used as part of staff training to highlight environmental contamination and to validate operator techniques.
Modifications to the method, such as using membrane filtration for enumeration, rather than the pour plate method described, may be necessary to produce a frequent use, in-house model. Current guidelines for transferring items from storage into the controlled work zone recommend a double disinfection process.2 The data presented in this paper do not disprove this need as the work only addresses a single disinfection process. However, further work to validate the modified method and address the double disinfection issue is being carried out, together with a pilot study, within a Welsh hospital, to prepare a standard operating procedure for validation of aseptic disinfection transfer techniques and for staff training.
Thanks are due to the Welsh school of pharmacy, Cardiff, for support and use of its facilities.
Dr Hiom is all-Wales research and development pharmacist based at St Mary’s Pharmaceutical Unit, University Hospital of Wales and Llandough NHS Trust, Penarth, Vale of Glamorgan CF64 2QX
|1.Medicines Control Agency. Rules and guidance for pharmaceutical manufacturers and distributors. London: Stationery Office; 1997.|
|2.NHS Quality Control Committee. The quality assurance of aseptic preparation services (2nd ed). London: Department of Health; 1996.|
|3.Hiom S. Surface bioburden of 25ml ampoules of isosorbide dinitrate. Research and development report, No 3. Cardiff: Welsh Pharmaceutical Services, 1999.|
|4.Payne DN. Microbial ecology of the production process. In: Denyer SO, Baird RM (editors). Guide to microbiological control in pharmaceutics. London: Ellis Horwood; 1990. pp53-67.|