Identifying Unknown Microbiological Contaminants in Cannabis
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We know microbiological contaminants pose a potential risk to cannabis consumers. In particular, bacteria and fungi may cause opportunistic infections immunocompromised individuals and even dead or dormant organisms may present a threat. As regulations have evolved across the US and Canada, it has become clear that robust, routine microbiological testing is essential in protecting consumer safety for medical patients and recreational users alike. As things stand, a patchwork of different testing regulations mandate a range of testing requirements across the US, and discussions on the best approach to guaranteeing accurate, reliable results are ongoing.
To find out more about the challenges faced and the technology available to the industry, we spoke to Amrita Puri, a Field Marketing Specialist with Bio-Rad, which offers a range of validated molecular- and culture-based diagnostic tools for the detection of pathogens in cannabis and other products.
Jack Rudd (JR): Why is identifying unknown contaminants in cannabis an issue?
Amrita Puri (AP): Because cannabis is grown in the environment, it can be naturally contaminated with pesticides, mold, or bacteria. Microbiological contaminants such as bacteria can be present at very low levels, as low as 1 colony forming unit (CFU) per sample size and pose a serious risk to humans because they produce toxins. Contamination can occur at growth, harvest, extraction, or even storage. The only way to ensure cannabis is free of microbial contamination and safe for human consumption is to test both raw and finished products on a routine basis with a proven technology that has been validated by international accrediting bodies such as AOAC, AFNOR, NordVal and Health Canada.
JR: What kind of contaminants most commonly go unidentified or misidentified in cannabis testing?
AP: Microbiological contaminants such as Salmonella, Shiga toxin-producing E. coli, and Aspergillus spp. Can go unidentified or misidentified in cannabis if vetted methods are not used for testing. For example, because these microorganisms can survive at very low levels under extreme environmental conditions, resuscitating sub-lethally injured cells in a high nutrient medium is critical. Detection methods that do not require an enrichment step can result in false negative results. Enrichment of microbes from cannabis products must be a prerequisite for accurate detection.
Aspergillus spp. Can be cultured on agar media, but not only are they very slow growers, it is also extremely difficult to differentiate the four species (A. fumigatus, A. flavus, A. niger and A. terreus) morphologically on a plate, which can lead to misidentification. Bio-Rad is currently beta testing the iQ-Check Aspergillus spp. kit and expects to launch the multiplex assay this year. A molecular approach to detecting Aspergillus spp. is not only more sensitive, but it is also a more accurate way to detect the microorganism.
JR: What tools does Bio-Rad offer to aid identification and how do they compare to other commonly used methods?
AP: Bio-Rad offers molecular and culture-based diagnostic tools for the rapid detection of pathogens in cannabis and cannabis-infused products. These include:
- Bio-Rad’s iQ-Check kits, which are based on gene amplification and detection by the use of real-time PCR and ready-to-use reagents and are optimized for microbial detection and identification in cannabis. PCR technology offers sensitive, specific and quick turnaround of results. Complex matrices contain inhibitory compounds that can shut down a PCR reaction. Following years of dedicated research and development, we have designed an array of robust PCR master mix with the inclusion of an internal amplification control that eliminates the risk of false negative results.
- Bio-Rad’s CFX96 Touch Deep Well real-time PCR Detection System provides a powerful platform for high-throughput microbial testing applications - an essential complement to iQ-Check kits. Bio-Rad manufactures the instrument and provides top-notch technical support to its customers.
- The iQ-Check Prep PCR Automation solution is dedicated to running the complete range of iQ-Check PCR Test Kits for high-throughput microbial testing. Automating the workflow frees up technician time by allowing walk away sample processing.
- RAPID’Chromogenic. As a complete solution provider, we also offer enrichment media and RAPID’Chromogenic media for easy bacterial detection, identification and quality indicator enumeration. Media offers cost-effective labor-saving protocols to detect and enumerate microbes, quickly and thoroughly.
JR: What are the biggest challenges and demands in this form of analysis in the cannabis industry?
AP: The biggest challenge is the lack of standardized testing. Currently, contaminant testing regulations vary by state and are inconsistent. For example, states like California require testing for Aspergillus species, pathogenic E. coli, and Salmonella, while the state of New York requires an array of tests for Pseudomonas, Enterococcus, bile-tolerant gram-negative bacteria, Clostridium, Mucor, Penicillium, and thermophilic actinomycetes species. Additionally, there is a lack of consensus on the testing methods. New methods offered in this industry are validated internally or by customer labs, but not by regulatory agencies that follow stringent testing guidelines. While cannabis is a complex matrix, the challenges associated with testing for microbes are similar to that in the food industry. The iQ-Check real-time PCR method has a proven track record in the food industry spanning decades.
JR: What needs to be done to improve microbiological contaminant analysis in the cannabis industry in your opinion?
AP: In my opinion, method validations must be standardized to ensure accurate testing. Performance criteria and reference methods must be defined. Validation studies must be conducted at independent laboratories to compare performance of methods to defined reference methods, such as the USDA FSIS MLG, FDA BAM, ISO, or Health Canada’s Compendium of Analytical Methods).
Validation protocols must make use of inoculation of cannabis matrices with live organism - not spiked DNA - using stressed or unstressed cells depending on the type of matrix, as the method validation is not complete unless both extraction and detection for the assay are tested. To further test the accuracy of the assay, probability of detection (POD) analyses, inclusivity, exclusivity, lot-to-lot stability, and robustness studies must be included in the validation studies.
Amrita Puri, a Field Marketing Specialist at Bio-Rad, was speaking to Jack Rudd, Managing Editor at Analytical Cannabis.