The Shortfalls Of Biological Indicators For HPV Decontamination Validation

The Vapour phase hydrogen peroxide (VPHP) process that is used for decontamination validation, conventionally uses Biological Indicators (BIs) to confirm decontamination has been achieved.  This translates into a lengthy process (which can take up to weeks or even months) with highly detailed protocols to be executed that we believe are highly un-optimised, costly and risky. The problems generally come from two areas –  a lack of understanding how the vapour phase hydrogen peroxide process actually works and a lack of consistency in the biological indicators used to challenge the process.

In this article, we are going to focus on the shortfalls of biological indicators for HPV decontamination validation and challenge the conventional processes with a better, revolutionary alternative – the Enzyme Indicator (EI).


The Conventional BI Process Falls Short In This Modern Age

While we believe that nobody should “cut corners” when it comes to cycle development, the whole process that is currently used can be highly improved and optimised.

For example, the wait for results is another key area – waiting days for a result is simply not viable in today’s fast paced pharmaceutical and medical environments. Scientists and Quality Control Managers need results now – in minutes. Not days.

The unreliable performance of BI’s in “rogue” results proves a real issue when it comes to risk and having to perform multiple cycles – at a cost of course.

Smoke pattern studies and temperature/humidity studies don’t actively contribute to the subsequent siting of test BIs. This means that the choice of BI sites is basically intuitive. Yet another area of practice which seems outdated.

While the choice of BI sites proves intuitive, we still believe that data from chemical indicators (CIs) may be incredibly helpful in providing supporting data for cycle development. Due to the nature of CIs, which react almost instantaneously as the gassing cycle proceeds, the results can be reviewed immediately, and the next development cycle modified accordingly. And this can be an excellent precursor to then validating decontamination with Enzyme Indicators (EIs).

We know that practical VPHP cycle development generally consists of the following three phases:

  1. Reviewing Existing Information

By gathering available information that relates to the current cycle development, a lot of time can be saved from being wasted on brand new cycle developments every time. Since a lot of information already exists, there is little point in not using it to predict the cycle parameters accurately before the actual development takes place.

  1. Chemical Indicators (CIs)

Since CIs are very cheap, large numbers can be used. This would mean that comprehensive information on the circulation of the gas is provided thus the likelihood of the HPV (Hydrogen Peroxide Vapour) process to be effective in relation to BIs is increased.

  1. Biological Indicators (BIs)

In reality, this is usually the real test of the bio-decontamination process. The convention is that any HPV cycle has to show log 6 reductions of Geobacillus Stearothermophilus spores. It’s argued by Tim Coles, in the Clean Air & Containment Review, that this is excessive, and log 4 reductions could be sufficient in a correctly-cleaned isolator.

With BI’s however you need to consider whether to duplicate or triplicate the BIs at each site. This is due to the unreliability of BIs because the so-called “rogue” BIs can sometimes be found – those BIs that survive the HPV process – and their rates are usually between 0.3% and 5%.

The strategy for dealing with these “rogue” BIs currently is to triplicate the BIs used for each process. This means that each process costs significantly more than the other, less common, duplication strategy, which proves more practical in cycle development executions.


Enzyme Indicators Transform Conventional Process

Having reviewed the shortfalls of Biological Indicators we would like to propose another solution in place of BIs. Enzyme Indicators (EIs) are the future!

Over the last decade, studies on enzymes produced by highly thermophilic bacteria have being conducted extensively. One enzyme, in particular, the thermostable adenylate kinase (tAK) has the unique properties that could lead it to overtake the use of BIs in decontamination cycles.

The HUGE advantage of EIs is that they can, like CIs, be read immediately after the gassing cycle is complete. This means that if EIs are used in the place of BIs, the whole process would take significantly less time, cost and effort for better, more accruate results!

We propose that the future HPV decontamination validation should consist of two key phases –

  1. Chemical Indicators (CIs)

Studies would be conducted using CIs in order to get a general overview of the isolator, load pattern, and gas generator performance. Ideally, the CIs and their immediate results would give maximum support to the subsequent phase.

  1. Enzyme Indicators (EIs)

EIs would then be used to give a precise numeric value to the equivalent log reduction achieved.

This cycle would be quicker and easier to conduct than the conventional cycle that the pharma and medical industries are using now. So much so, that developments that currently take weeks or even months, can now be done in days.

Another significant benefit would be that the documentation and results would be much simpler and more comprehensible to everyone, regardless of technical expertise.


In conclusion, the current HPV decontamination validation cycles are conducted in a very expensive and lengthy manner which can be dramatically shortened and made cheaper by our proposed process of using EIs. We believe that the future is here and this can be one of the most revolutionary ways to transform decontamination validation as we know it.


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