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!