The need to be able to verify if cleaning and disinfection
procedures have been completed to ensure successful eradication of pathogens,
particularly in outbreak settings, is of significant importance. In the Healthcare market we have seen a rapid
uptick in the use of UV markers or ATP.
As mentioned last week, 2015 will include topics of interest in the
Animal Health market and not surprisingly, they have their own way of
validating cleaning and disinfection practices.
Where ATP, UV Reflectance or environmental swabbing are commonly used in
the Healthcare sector, the Animal Health sector seems to favour Polymerase
Chain Reaction (PCR) for determining the health status of animals, as well as
for validating cleaning and disinfection practices. However, as identified in previous blogs, ATP
cannot be used to compare different disinfectant technologies and that caution
needs to be used when interpreting results. Similar to the impacts of
different disinfectants on ATP tests, it is important to recognize that the
mechanism of action of disinfectant chemistries can have a direct impact on the
use and interpretation of PCR test results.
PCR is a biochemical method in which a single strand of DNA
can be multiplied into thousands and millions of exact copies. In a way, PCR is
a “photocopier” of genetic materials. The reaction often takes place in a
single vial where the necessary reagents (polymerases, primers, nucleic acids,
buffers, etc.) are combined into a single solution. The vial is placed in a
machine which can be programmed to rapidly take the solution through various
cycles of temperature (a PCR machine). It is through this calculated
temperature cycling of the solution that the enzymes exponentially produce new
copies of the original DNA strand.
The use of PCR is great if one is trying to selectively
amplify a known segment of an organism’s DNA. The organism can be a bacteria,
virus, or a mammalian cell. The PCR method is extremely selective (due to the
specific primer molecule designs), and therefore it will only amplify the
targeted strand of DNA. This means that, if one was looking to amplify a
segment of poliovirus DNA for example, the solution in the vial must have at
least one copy of that target DNA or at least have one poliovirus cell.
The key to understanding the limitations for using PCR as a
validating tool for cleaning and disinfection is the fact that it can amplify
DNA molecules regardless of the origin of the DNA. The origin of the DNA could
be from another PCR run, a live bacterial cell, an infective virus cell, or a
ruptured cell content. Therefore, PCR cannot be used to confirm if a surface
has been disinfected, as PCR is not designed to check for virulence and infectivity
of various pathogens and is only used to amplify a target DNA. The DNA can come
from live or dead pathogen.
In general,
disinfectants are designed to inactivate pathogenic agents and to ensure
microbes no longer have the ability to be infective. There are many different
active ingredients that are used in formulating disinfectant products, each
with a distinct mechanism of action. In other words, each disinfectant active
ingredient has its own way to inactivate the pathogens. Some block metabolic
centers of the bacteria, leading to bacterial cell death, others may rupture
the cell membrane of the bacteria or virus. Other active ingredients may target
and degrade the genetic material of the pathogenic cells, and others may
deactivate these cells by degrading the functional cell enzymes. Regardless of
the method, in all the examples above the pathogens are inactivated so that
they no longer are alive or are able to cause infection. Using PCR to test
disinfection of a surface can therefore be misleading as one active ingredient
might kill pathogens through rupturing their cell membrane, while not degrading
the DNA within the cell. This would cause a false positive when using PCR, as the
presence of DNA of a pathogen on the surface by no means indicates it is still
infectious!
Bugging Off!
Nicole
