Not as easy as 1-2-3: ATP swabbing and its relevance for controlled environments

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Updated: Oct 9

Technical Review Article | Open Access | Published 11th October 2024

Not as easy as 1-2-3: ATP swabbing and its relevance for controlled environments

Tim Sandle, Ph.D., CBiol, FIScT | EJPPS | 293 (2024) | Click to download pdf

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For non-aseptic areas, the adenosine triphosphate (ATP) bioluminescence assay, based on the measurement of levels of ATP present on an environmental surface, presents a potential alternative method to culture-based bioburden determinations. ATP is the principal energy carrier for all living organisms and hence it is a potential indicator.

The bioluminescence test utilizes the chemiluminescence properties of a luciferin-luciferase reagent, which reacts with any ATP residue present on a substrate, emitting light and measuring the presence of organic matter ¹.

The advantage of ATP-swabbing is that the level of training is lower, data can be obtained closer to real-time, and results can be provided within the manufacturing environment. The technology is also relatively well-established within sectors like the food industry.

But how accurate is the ATP assay and how much can we rely on the data? This article looks at how the ATP assay works and considers the advantages and disadvantages of the approach.

ATP swabs. Image by Tim Sandle

Description of the method

The ATP bioluminescence method comprises of a swab, used to sample a standardized area using the conventional swabbing technique. The swab is subsequently placed in a tool that uses the firefly genus Photinus) enzyme luciferase to catalyze the conversion of ATP into Adenosine Monophosphate (AMP). This chemical reaction results into an emission of light which is then detected by a bioluminometer. The light emitted during the reaction was directly proportional to the amount of ATP.

A firefly. Image by Tim Sandle

Specifically, in the presence of oxygen and magnesium ions, luciferin is converted to oxyluciferin, and ATP is converted to AMP with the release of pyrophosphate and the emission of light in the wavelength range of 470–700 nm.

The obtained results are in the form of ‘relative light units’ (RLU) expressed per the area of surface measured. These luminometric are probabilistic values in relation to suspected levels of microbial contamination.

Swabbing method

The method of swabbing is important and undertaking this consistently has an important bearing on compare results over time and for laboratory-to-laboratory comparisons. An example technique would be swabbing first in one direction and then in the opposite direction. A suitable sample area might be area of 10 × 10 cm. In terms of selecting the type of swab, the chosen swab needs to be certified to be free of ATP to eliminate the risk of cross-contamination. Protecting the sampled swab from cross-contamination up to the point of testing is also of importance.

Capturing the light. Image by Tim Sandle

Depending on the swab and instrumentation, ATP testing can detect as little as 5 picograms 1/10000000000 grams) of organic material.

Advantages

It is simple.
The method is relatively sensitive.
Cost effective.
Rapid (compared to conventional providing real-time results within minutes.
While there is no accepted benchmark, for a non-sterile environment a reading of more than 100 RLU/100cm^2 is regarded as representing a ‘high’ value and a surface with a potential bioburden that is unacceptable for pharmaceutical production (as well as general healthcare tasks) ².

Disadvantages

There are several disadvantages with the ATP method, not least a relatively wide variability ³. These disadvantages do not necessary negate the use of the method, but they do need consideration when designing a sampling regime and for results interpretation:

ATP is an indicator of organic material presence, rather than microbial contamination ⁴.
The signal from the method does not necessarily signal viable bacteria for surfaces include remnants of dead bacteria.
The method is not very sensitive for spore detection.
There is no direct correlation between assay reading and microbial numbers ⁵.
The method cannot differentiate between microbial species.
ATP has a short half-life; therefore testing must be done shortly after sampling.
Cellular enzymes quickly degrade ATP.
The assay is poorly standardized and there are currently no widely accepted benchmarks.
The method is affected by different surface materials ⁶.

There are some other things that can affect the method, including disinfectant residues (especially the presence of chlorine which can interfere with the assay) ⁷. Another source of interference can be fibres shed from cleaning materials ⁸.

Cleanroom life. Image by Tim Sandle

Correlation with viable counts

What does the assay tell us about levels of bioburden? ⁹ While a number of studies present strong evidence for the existence of such a correlation ¹⁰, in my experience the correlation is not clear cut. Moreover, many studies have found no statistical correlation was found between ATP values and viable counts ¹¹. However, both ATP swabbing and viable counts were consistent in indicating the general evaluation of hygienic quality of surfaces. In addition, TP mean values showed a progressive increase from surfaces as the bioburden increased ¹².

Instrument reliability

As to how reliable the instruments are, a large study found considerable variability of with four ATP bioluminometers and their consumables, upwards of 50%. The authors considered this to be unacceptably high (although it was within the manufacturers’ technical configuration). The instrument imprecision was most apparent with the numerical scale of RLUs ¹³.

Because each manufacturer uses a different scale, RLU values should not be used to compare other ATP monitoring systems.

The art of swabbing. Image by Tim Sandle.

More recent advances have seen the introduction of biosensors based on photodiode arrays ¹⁴.

Test variability

ATP tests can be skewed by sampling and testing variables. One example is wet surfaces – a test taken in wet conditions will be liable to be higher than the actual cleanliness of the surface.

Setting a limit

Based on the above, setting an RLU value will be difficult. However, if ATP-swabs and conventional bioburden monitoring (such as using contact plates or flocked swabs) is performed over a reasonable period of time as so to generate a sufficient number of samples (such as producing 100 comparative sampled over three-months), the n based on the relationship between RLU values and bioburden recoveries, then an internal target value corresponding to the 75 percentile of the RLU detected values could be set to function as an action level. From this, an internal alert level can be set at a suitable deviation from the action level, such as 20% below ¹⁵.

Bacterial culture. Image by Tim Sandle

Other applications

ATP testing can additionally be used to test water quality in a variety of different settings. This can be especially useful following an incident, where a quick assessment of the effectiveness of any remediation activity is required.

What can we conclude?

Based on the above, and accounting for other information, what can we conclude about the suitability of the ATP method?

ATP bioluminescence is not an alternative to cultural methods.
However, the ATP-bioluminescence-assay can be a useful tool to measure the efficiency of cleaning procedures in areas where a general cleanliness check is required.
By providing rapid feedback, the ATP-assay can increase the awareness of operators and adverse results can enable immediate action to be taken in critical situations.
Each health facility should seek to identify an appropriate reference value.
The reference value is not absolute, it will vary according to device type and data collection and analysis.
Sample results will vary, as with any microbiological monitoring, according to spatial and temporal factors and according to the type of surface sampled (as well as the adhesion and detachment of any microorganisms bound to the surface).

Therefore, how effective is ATP as a proxy of microbial contamination? The answer is a little vague and uncertain and hence the method is best reserved as a crude approximator of the level cleanliness rather than an absolute indicator of surface microbial acceptability.

References

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