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Testing & Processing Mycotoxin analysis

Culture Shock

Cannabis is used to treat a huge variety of conditions, some of which are immunocompromising, so it is of paramount importance to ensure that products are free of microbial contaminants.

At present, microbial testing requirements for cannabis differ widely from state to state – some call for molecular methods while others rely on culture-based technologies such as plating. Some regulations require total counts, while others focus on known human pathogens, and others require a mixture of the two. Testing methods used in food are commonly being adapted to cannabis; however, cannabis is a unique matrix in itself and has myriad alternative matrices (for instance, concentrates and infused products), which further complicates the picture.

In 2015, the Medicinal Genomics team sequenced the cannabis microbiome of a variety of cultivars, revealing the presence of numerous mycotoxic fungi in dispensary-grade cannabis (1). Further work has demonstrated that many of these pathogenic fungi are endophytes, meaning that they reside inside the cannabis plant (unlike epiphytes, which colonize the surface of the plant) (2, 3). Some of these pathogens include Aspergillus, Fusarium, and Rhizopus (4), all of which produce mycotoxins and have been implicated in a number of cases in which people became ill ­­– or even died – after using cannabis, with immunocompromised patients most at risk (5,6,7,8,9,10,11).

Endophytes are a major blind spot for culture-based systems. The methods used to collect samples for culture are predominantly designed to pick up surface microbes, and typically only capture a very small proportion of endophytic communities.

Furthermore, some microorganisms of concern, like Aspergillus, do not grow well in culture-based systems and have a propensity to clump and produce macrocolonies, making the standard colony-forming unit an inaccurate measure. To further complicate the picture, different species of Aspergillus can be morphologically very similar, making the distinction between what is pathogenic and not extremely challenging. In one instance, the state of Alaska had to step in to referee a disagreement between two labs over misidentification of A. niger (pathogenic) for A. brasiliensis(non-pathogenic).

The Medicinal Genomics team has also observed a large discordance between different culture-based platforms, as well as how these two platforms compare to quantitative polymerase chain reaction (qPCR), a DNA-based test. In a follow-up study, our team demonstrated that the act of culturing produces a skewed image of the cannabis microbiome and that many of culture-based systems lack specificity and grow off-target microorganisms, leading to inflated total counts (12). Furthermore, there were many instances when qPCR would yield signal while the plates were clean and vice versa. Sequencing of the colonies on these plates and the amplicons generated from qPCR demonstrated the presence of an endofungal bacteria called Ralstonia. This is problematic for non-molecular methods because these bacteria take residence inside of fungal cells and therefore the cell must be lysed to know if they are present. Ralstonia is a pathogen to both plants and humans, causing wilt in the former and lung infections in the latter (13,14).

Even if a product appears to be free of viable contaminants according to culture-based techniques, many known endophytic and endofungal pathogens could be missed. Plus, there is no good way to homogenize samples without lysing cells. Grinding will lyse cells in a non-uniform manner and anything that lyses a plant cell could also lyse a microbial cell. Therefore, relying on techniques that only measure viable cells can lead to increased failure rates for products that are perfectly safe while failing to detect underlying microbial threats.

There are numerous challenges that face the accurate quantification of microbial hazards in cannabis; in my view, molecular methods are best able to address them and ensure consumer safety.

While total count tests can give you some information about the microbial load in a sample they lack specificity and do not differentiate between what is hazardous and what is benign, which puts growers who use beneficial microbes in a tricky spot. This is why I am a strong advocate for species-specific testing. Targetings known threats is a much better way of ensuring a product is safe without penalizing cultivators for organic practices. Carefully designed qPCR primers are one way to resolve this issue of specificity.  Some industries under the jurisdiction of the US Food and Drug Administration are taking things a step further and are beginning to sequence contaminated product to better understand the exact serotype of the pathogens encountered during outbreaks.

As we begin to shape policy for newly legal states – and eventually federal legalization – I hope that regulators take these important factors into consideration. Ultimately, the safety of consumers relies on implementing scientifically sound approaches.

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  1. K Mckernan et al., “Cannabis microbiome sequencing reveals several mycotoxic fungi native to dispensary grade Cannabis flowers”, F1000Re, 4, 1422 (2015). DOI: 10.12688/f1000research.7507.2.
  2. AK Gautam et al., “Isolation of endophytic fungi from Cannabis sativa and study their antifungal potential”, Arch Phytopathology Plant Protect, 46, 627–635 (2013). DOI: 10.1080/03235408.2012.749696
  3. P Kusari et al., “Endophytic fungi harbored in Cannabis sativa L.: diversity and potential as biocontrol agents against host plant-specific phytopathogens”, Fungal Divers, 60, 137–151 (2012). DOI:10.1007/s13225-012-0216-3. 
  4. M Scott et al., “Endophytes of industrial hemp (Cannabis sativa L.) cultivars: identification of culturable bacteria and fungi in leaves, petioles, and seeds”, Can J Microbiol, 64, 664–680 (2018). DOI: 10.1139/cjm-2018-0108.
  5. Y Gargani et al., “Too many mouldy joints - marijuana and chronic pulmonary aspergillosis”, Mediterr J Hematol Infect Dis, 3, e2011005 (2011). DOI: 10.4084/MJHID.2011.005.
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  7. M Szyper-Kravitz et al., “Early invasive pulmonary aspergillosis in a leukemia patient linked to aspergillus contaminated marijuana smoking”, Leuk Lymphoma, 42, 1433–1437 (2001). DOI: 10.3109/10428190109097776.
  8. WH Marks et al, “Successfully treated invasive pulmonary aspergillosis associated with smoking marijuana in a renal transplant recipient”, Transplantation, 61, 1771–1774 (1996). DOI: 10.1097/00007890-199606270-00018.
  9. R Llamas et al., “Allergic bronchopulmonary aspergillosis associated with smoking moldy marihuana”, Chest, 73, 871–872 (1978). DOI: 10.1378/chest.73.6.871.
  10. R Hamadeh et al., “Fatal aspergillosis associated with smoking contaminated marijuana, in a marrow transplant recipient”, Chest, 94, 432–433 (1988). DOI: 10.1378/chest.94.2.432.
  11. T Stone et al, “Pulmonary mucormycosis associated with medical marijuana use”, Respir Med Case Rep26, 176–179 (2019). DOI: 10.1016/j.rmcr.2019.01.008.
  12. K Mckernan et al., “Metagenomic analysis of medicinal samples; pathogenic bacteria, toxigenic fungi, and beneficial microbes grow in culture-based yeast and mold tests”, F1000Res, 5, 2471 (2016). DOI: 10.12688/f1000research.9662.1.
  13. T Coenye et al., “Infection by Ralstonia species in cystic fibrosis patients: identification of R. pickettii and R. mannitolilytica by polymerase chain reaction”, Emerg Infect Dis, 8, 692–696 (2002). DOI: 10.3201/eid0807.010472.
  14. N Peeters, “Ralstonia solanacearum, a widespread bacterial plant pathogen in the post-genomic era”, Mol Plant Pathol, 14, 651–662 (2013). DOI: 10.1111/mpp.12038.

About the Author

Kyle Boyar

Field Application Scientist, Medicinal Genomics, Beverly, Massachusetts, USA.

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