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YOU’VE GOT TO FIND THE BUGS: A CONVERSATION WITH NORTH COAST TESTING LABORATORIES ABOUT MICROBIAL CONTAMINATION

The madness that we’re all currently enduring as we live the oxymoron of social distancing harkens to the fact that microorganisms can be deadly. At the very least, they might cause illnesses. A 2018 study claimed that a medical cannabis patient contracted meningitis due to inhaling cannabis tainted with Cryptococcus neoformans. [1] Other studies have associated aspergillosis with long-term cannabis smoking. [2] Regardless of these findings, the need for uncontaminated cannabis should go without saying, shouldn’t it, especially when the word medical is added to the conversation?

I recently spoke with Adam Scavone, of North Coast Testing Laboratories located outside of Cleveland, Ohio. Scavone sees room for improvement in several states’ regulations, including Ohio and Illinois, regarding microbial testing, as well as the analytical tests themselves.

“We strongly believe in testing, especially for dangerous yeasts and molds,” Scavone began. “However, a particular test, commonly required by regulation in cannabis and conventional drugs, known as the ‘Total Yeast & Mold Count’ [TYMC] is not adequate for screening cannabis products that are commonly used via inhalation, especially flower and solventless concentrates. Regulations need to require screening these products with intensive, sensitive, and reliable methods for specific, dangerous organisms to offer the greatest degree of protection to patients.”

Any damaging global crisis like the current pandemic is bound to spark the fire in others to extract some education from the experience – what can we be doing differently? Our solutions may not squash coronavirus, per se, but our ability and desire to change how we’ve been doing things can motivate us onto superior paths.

“When the COVID-19 outbreak hit our radar screen and the potential tsunami facing Ohio’s healthcare system became apparent, we looked for ways to help,” Scavone explained. “With a pandemic all around us, and with the vape issues of last year burned into our recent memory, our top priority quickly became taking a closer look at respiratory health risks faced by cannabis patients.”

With that focus on respiration, Scavone and his colleagues pointed to an “obvious and relatively easy win”: screening cannabis flower and solventless extracts for Aspergillus species, including A. flavus, A. fumigatus, A. niger, and A. terreus, which pose a threat to human health, especially respiratory health.

They first consulted the scientific literature and found the 2011 report that showed a photograph of a nine centimeter-wide “fungal ball” of A. fumigatus that had grown in a patient’s lungs, apparently after consistently inhaling the fungus daily from a supply of contaminated, black-market cannabis. [2] A 2017 report documented the widespread presence of pathogenic Aspergillus species in cannabis purchased in California dispensaries, prior to mandated testing. [3] The literature validated the credence of focusing on better analytics.

The regulatory problem is this: Ohio’s regulations allow medical cannabis flower with 9,999 colony-forming units (CFU) of A. fumigatus per gram to “pass” because it’s under the 10,000 CFU per gram threshold for the TYMC test. This test was adopted from the American Herbal Pharmacopoeia’s Cannabis Inflorescence monograph published in 2014.

“The TYMC test is dangerously deceptive,” Scavone warned. “As a standalone test, without additional pathogen-specific testing requirements, patients, caregivers, dispensary operators, and even cultivators and processors with the best intentions are given a false sense of product safety.”

Scavone said that adopted microbial tests during this timeframe didn’t really consider quantitative polymerase chain reaction (qPCR) since it wasn’t “economically feasible for cannabis testing labs.” He believes qPCR provides a more effective assessment of microbial contamination. Most recently, qPCR has become the trusted detection method for identifying COVID-19 infection.

In qPCR, cannabis samples are extracted into liquids whereby all DNA helixes that exist in the sample are captured including that from dangerous microorganisms, plant material, and any other organisms growing on the plant.

The DNA is then denatured (split into two strands) by heat. An enzyme – the polymerase – is added, along with the building blocks of DNA (adenine, cytosine, guanine, and thymine, ACGT) and a fluorescent dye that specifically binds to the “target organism” DNA. The split DNA strands are paired with ACGT and, critically, the fluorescent dye, resulting in two dyed copies. The cycle repeats, resulting in four dyed copies, then eight, and so on.

“The selectivity comes from primers having complementary DNA pairs to the DNA sequence of the target organism,” Scavone explained. “A binds to T and G binds to C, so the primer itself is a perfect complementary pair to the target DNA sequence and the primer cannot bind to any other DNA sequence that doesn’t complementarily match its sequence.”

After enough cycles of denaturing and replication, if the target organism is present, its dyed DNA strands become fluorescent and visible on qPCR instruments, which look for the specific fluorescence spectrum that corresponds to the dye.

“By tracking the number of quantitation cycles that occur before the fluorescence becomes visible,” Scavone explained, “we know the number of organisms that were present at the start of the process – giving us the “quantitative” component of qPCR.”

“qPCR is superior,” he continued, “since it can specifically and accurately determine whether a cannabis sample contains dangerous organisms, providing a significant advantage over culture-based methods. In technical terms, qPCR is also highly sensitive – if a target organism exists in the tested sample, it’s nearly certain to be identified by qPCR.”

In comparison, Scavone pointed to a major drawback of culture-based methods: they may not grow the target organism, even if it’s thriving in the cannabis sample. “In our own lab,” he said, “we have demonstrated inhibition of Salmonella growth from a “spiked” sample by competition from Citrobacter, a non-toxic but competitive bacteria that ‘eats’ the nutrients on the plate before the Salmonella can.”

This results in the false notion that no Salmonella exists in the sample, a false negative, when, in fact, the bacteria is present.

Since qPCR measures DNA, it can identify the living and the dead. If the organisms are dead, they’re no longer proliferating or creating mycotoxins. But even if the DNA of dead organisms is detected, some cultivators may view this as valuable data, since, at one point in time, their crop was contaminated.

“The first question that every cultivator and processor needs to ask is whether they actually want to discriminate between dead and live organisms,” Scavone commented. “If there was living Aspergillus on the batch or lot, but now it’s dead, some cultivators and processors will view that as a red flag, even though under some states’ laws, it would “pass” presumably on the theory that dead Aspergillus lack the capability to cause aspergillosis or any kind of infection.”

There is a clean-up procedure to remove DNA from dead organisms, leaving the living organism DNA. This technique involves mixing the sample with a reagent that can only penetrate cell membranes of dead cells. The reagent passes through the cell membranes of dead cells and binds to the DNA inside them when exposed to bright light. This binding means that the dead cells’ DNA cannot bind with the fluorescent nucleotides (ACGT) added in the “amplification” procedure. No fluorescence means no detection by the instrument.

The take-home message of this conversation isn’t rooted in ominous forewarning that the cannabis you use every day is contaminating you. The endgame of more rigorous microbial testing methods is always consumer protection.

“To be very clear, Scavone concluded, “the occurrence of Aspergillus species with toxic potential in modern, well-regulated, commercial cannabis cultivation and processing systems is extremely rare. Our goal is prevention and reducing the risk of aspergillosis to zero. Over time, and with enough volume, the odds greatly increase that contamination will occur, simply by random chance. We want to ensure we do everything in our power to catch contamination before products arrive at dispensaries. As the current COVID-19 crisis has taught us all, we all need to be vigilant and hyperaware of keeping people safe and healthy.”

References

  1. Shapiro, B. et al. “Cryptococcal Meningitis in a Daily Cannabis Smoker without Evidence of Immunodeficiency,” BMJ Case Reports, v. 2018, 2018, bcr2017221435. [journal impact factor = ; timed cited ]
  2. Gargani Y et al. “Too Many Mouldy Joints – Marijuana and Chronic Pulmonary Aspergillosis. Mediterr J Hematol Infect Dis, vol. 3, no. 1, 2011, pp. e2011005. [journal impact factor = ; timed cited ]
  3. Thompson III, G. et al. “A Microbiome Assessment of Medical Marijuana,” Clinical Microbiology and Infection, vol. 23, no. 4, 2017, pp. 269-270. [journal impact factor = ; timed cited ]

About the author

Jason S. Lupoi, Ph.D.

Jason S. Lupoi, Ph.D.

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