Techniques and Technologies for Microbiological Contamination Testing
The conidiophore of the fungal organism Aspergillus fumigatus. Obtained from the CDC Public Health Image Library. Credit: CDC/Dr. Libero Ajello (PHIL #4297),
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Having access to effective and accurate methods for cannabis testing is imperative to ensuring the safety of medicinal and recreational cannabis users. The American Herbal Products Association and the American Herbal Pharmacopoeia both issue guidelines on how to conduct cannabis cultivation, processing, and dispensing in order to minimize risks from contamination, but without proper testing methods, it is impossible to quantify the risk presented by contamination.
In the absence of any federal safety regulations and procedures from either the FDA or the USDA, it is up to individual states to implement their own cannabis testing procedures. Two of the most common microbiological testing methods used are traditional culture methods (TCM), and quantitative polymerase-chain-reaction (qPCR) analysis.
Common Microbiological Testing Methods
Culture-based microbial testing is considered the by many as the go-to standard for detecting harmful microbes. TCM is a useful technique as it is relatively simple and straightforward to carry out and it is possible to purchase sample-ready culture medium systems specifically designed for common processes such as total yeast and mold tests. Recently, qPCR has also gained prevalence as a more targeted approach to testing. qPCR works by amplifying sections of DNA or RNA present in the sample that are characteristic to the compounds or microbes that are being tested for. The use of qPCR has become more common as foodstuffs and biological materials with short shelf lives are often unable to be analyzed using TCM and as such a more rapid testing approach is required.
As cannabis testing is not federally regulated, states are free to mandate the use either of these analytical methods to examine cannabis samples. Each method comes with its own pros and cons that need to be considered to better understand their suitability and compatibility.
A Comparison of TCM and qPCR
One of the biggest advantages that qPCR has over TCM is the speed at which results are produced. Carrying out TCM can take between 24-48 hours depending on the time required to grow and analyze the culture. In comparison, qPCR runs often last only two hours from start to finish and can be done in as little as 40 minutes on some instruments.
Both methods do require a certain amount of experience in order to interpret their respective outputs. Some TCM plates are very easy to interpret but in cultures where there may be more than one type of bacterium or fungus present, it can require an experienced eye to differentiate between the species and draw accurate conclusions. Interpretation of qPCR results was similarly difficult until recently, but with the creation of genomic databases of sequences found from previous runs, it is getting easier.
Another factor to bear in mind with qPCR is that even though a DNA sequence may be detected it may not actually be expressed. For example, in one study genes for the bacterial and fungal toxins, paxilline and citrinin respectively were identified in a cannabis sample via qPCR analysis. However, follow up analysis using a mass spectrometry-based method found no evidence of the presence of either of these toxins. This finding presents two different potential challenges.
1. The genes are not being expressed and therefore no toxin is being produced but, based on qPCR analysis alone the sample could fail testing as it is considered contaminated.
2. The sample used for MS analysis may not be representative of the cannabis batch in question and the toxins may be present elsewhere on the plant material
Either way, this case highlights the need for careful testing to ensure any given cannabis product does not present a clinical risk to users. This is discussed in a talk titled “Cannabis Microbiome Sequencing: Impact on Microbial Safety Testing” by Kyle Boyar, Medicinal Genomics.
Similarly, TCM is not a perfect method. Many types of microbe do not culture well, and so will be underrepresented by this method. This can have potentially dangerous consequences when the bacteria being underrepresented are dangerous to humans. A prime example of this is the Aspergillus species. Spores from Aspergillus molds can be dangerous to people who are immunocompromised as the inhalation of these spores can lead to aggressive pulmonary diseases. As a result of this danger, it is one of the primary contaminants that is screened for during cannabis quality control. Aspergillus does not culture well on some of the most common TCM media, leading its presence to be underreported. This can create a feedback loop where since it is known that Aspergillus is not easily detected, more antimicrobials and fungicides are used on the cannabis crop in an attempt to preemptively curb the Aspergillus risk. However, many of these fungicides are dangerous contaminants in their own right as they can act as endocrine disruptors or neurotoxins in the human body, so their increased usage simply presents new risks.
Aspergillus is not the only species that is misrepresented using TCM analysis. Culture-based analysis on the whole struggles to distinguish between fungi and bacteria colonies, which can distort the final colony count of the culture and create false-positive readings when determining the microbiome of plant material. qPCR analysis is able to distinguish between fungi and bacteria colonies by using characteristic DNA primers, and so returns far fewer false-positive readings.
Due to its ease of use and general accessibility, qPCR is growing in popularity as a method for microbiological testing. It is able to detect specific microbial risks through DNA analysis and does so with less distortion to the final results, hence creating a far more accurate picture of the microbial environment present on the cannabis material. The confidence with which qPCR can be used also offers a way to halt the destructive feedback loop that leads to raised fungicide and pesticide use, in turn protecting the environment on which the pesticides are used and also any farm workers or cannabis users who come into contact with the treated cannabis.
These findings also have implications outside the cannabis industry, as TCM is still one of the major methods being to monitor the quality and safety of other foodstuffs. Just as it has been shown to be insensitive to the Aspergillus species, there is a potential concern that it may be insensitive to other fungal or bacterial infections that may be hosted on foodstuffs. Recognizing this, it is important to focus on using our increased understanding of crop microbiomes across industries to improve cultivation, handling, and processing methods.