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Rapid Pathogen Detection for the 21st Century: A Look at PathogenDx

By Aaron G. Biros
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In 1887, Julius Petri invented a couple of glass dishes, designed to grow bacteria in a reproducible, consistent environment. The Petri dish, as it came to be known, birthed the scientific practice of agar cultures, allowing scientists to study bacteria and viruses. The field of microbiology was able to flourish with this handy new tool. The Petri dish, along with advancements in our understanding of microbiology, later developed into the modern field of microbial testing, allowing scientists to understand and measure microbial colonies to detect harmful pathogens in our food and water, like E. coli and Salmonella, for example.

The global food supply chain moves much faster today than it did in the late 19th century. According to Milan Patel, CEO of PathogenDx, this calls for something a little quicker. “Traditional microbial testing is tedious and lengthy,” says Patel. “We need 21st century pathogen detection solutions.”

Milan Patel first joined the parent company of PathogenDx back in 2012, when they were more focused on clinical diagnostics. “The company was predominantly built on grant funding [a $12 million grant from the National Institute of Health] and focused on a niche market that was very specialized and small in terms of market size and opportunity,” says Patel. “I realized that the technology had a much greater opportunity in a larger market.”

Milan Patel, CEO of PathogenDx
Photo: Michael Chansley

He thought that other markets could benefit from that technology greatly, so the parent company licensed the technology and that is how PathogenDx was formed. Him and his team wanted to bring the product to market without having to obtain FDA regulatory approval, so they looked to the cannabis market. “What we realized was we were solving a ‘massive’ bottleneck issue where the microbial test was the ‘longest test’ out of all the tests required in that industry, taking 3-6 days,” says Patel. “We ultimately realized that this challenge was endemic in every market – food, agriculture, water, etc. – and that the world was using a 140-year-old solution in the form of petri dish testing for microbial organisms to address challenges of industries and markets demanding faster turnaround of results, better accuracy, and lower cost- and that is the technology PathogenDx has invented and developed.”

While originally a spinoff technology designed for clinical diagnostics, they deployed the technology in cannabis testing labs early on. The purpose was to simplify the process of testing in an easy approach, with an ultra-low cost and higher throughput. Their technology delivers microbial results in less than 6 hours compared to 24-36 hours for next best option.

The PathogenDx Microarray

Out of all the tests performed in a licensed cannabis testing laboratory, microbial tests are the longest, sometimes taking up to a few days. “Other tests in the laboratory can usually be done in 2-4 hours, so growers would never get their microbial testing results on time,” says Patel. “We developed this technology that gets results in 6 hours. The FDA has never seen something like this. It is a very disruptive technology.”

When it comes to microbial contamination, timing is everything. “By the time Petri dish results are in, the supply chain is already in motion and products are moving downstream to distributors and retailers,” Patel says. “With a 6-hour turnaround time, we can identify where exactly in the supply chain contaminant is occurring and spreading.”

The technology is easy to use for a lab technician, which allows for a standard process on one platform that is accurate, consistent and reproduceable. The technology can deliver results with essentially just 12 steps:

  1. Take 1 gram of cannabis flower or non-flower sample. Or take environmental swab
  2. Drop sample in solution. Swab should already be in solution
  3. Vortex
  4. Transfer 1ml of solution into 1.5ml tube

    A look at how the sample is added to the microarray
  5. Conduct two 3-minute centrifugation steps to separate leaf material, free-floating DNA and create a small pellet with live cells
  6. Conduct cell lysis by adding digestion buffer to sample on heat blocks for 1 hour
  7. Conduct Loci enhancement PCR of sample for 1 hour
  8. Conduct Labelling PCR which essentially attaches a fluorescent tag on the analyte DNA for 1 hour
  9. Pipette into the Multiplex microarray well where hybridization of sample to probes for 30 minutes
  10. Conduct wash cycle for 15 minutes
  11. Dry and image the slide in imager
  12. The imager will create a TIFF file where software will analyze and deliver results and a report

Their DetectX product can test for a number of pathogens in parallel in the same sample at the same time down to 1 colony forming unit (CFU) per gram. For bacteria, the bacterial kit can detect E. coli, E. coli/Shigella spp., Salmonella enterica, Listeria and Staph aureus, Stec 1 and Stec 2 E.coli. For yeast and mold, the fungal kit can test for Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger and Aspergillus terreus.

Their QuantX is the world’s first and only multiplex quantification microarray product that can quantify the microbial contamination load for key organisms such as total aerobic bacteria, total yeast & mold, bile tolerant gram negative, total coliform and total Enterobacteriaceae over a dynamic range from 100 CFU/mL up to 1,000,000 CFU/mL.

Not all of the PathogenDx technology is designed for just microbial testing of cannabis or food products. Their EnviroX technology is designed to help growers, processors or producers across any industry identify areas of microbial contamination, being used as a tool for quality assurance and hazard analysis. They conducted industry-wide surveys of the pathogens that are creating problems for cultivators and came up with a list of more than 50 bacterial and fungal pathogens that the EnviroX assay can test for to help growers identify contamination hotspots in their facilities.

Using the EnviroX assay, growers can swab surfaces like vents, fans, racks, workbenches and other potential areas of contamination where plants come in contact. This helps growers identify potential areas of contamination and remediate those locations. Patel says the tool could help growers employ more efficient standard operating procedures with sanitation and sterilization, reducing the facility’s incidence of pathogens winding up on crops, as well as reduction in use of pesticides and fungicides on the product.

Deploying this technology in the cannabis industry allowed Milan Patel and the PathogenDx team to bring something new to the world of microbial testing. Their products are now in more than 90 laboratories throughout the country. The success of this technology provides another shining example of how the cannabis market produces innovative and disruptive ideas that have a major impact on the world, far beyond cannabis itself.

3 Essential Components of Microbial Safety Testing

By Heather Ebling
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Microbial contamination on cannabis products represents one of the most significant threats to cannabis consumers, particularly immunocompromised patients who are at risk of developing harmful and potentially fatal infections.

As a result, regulatory bodies in the United States and Canada mandate testing cannabis products for certain microbes. The two most popular methods for microbial safety testing in the cannabis industry are culture-based testing and quantitative polymerase chain reaction (qPCR).

When considering patient safety, labs should choose a method that provides an accurate account of what is living on the sample and can specifically target the most harmful microbes, regardless of the matrix.

1. The Method’s Results Must Accurately Reflect the Microbial Population on the Sample

The main objective of any microbial safety test is to give the operator an indication of the microbial population present on the sample.

Figure 1: MA data collected directly from plant material before and after culture on 3M petrifilm and culture-based platforms.

Culture-based methods measure contamination by observing how many organisms grow in a given medium. However, not all microbial organisms grow at the same rate. In some cases, certain organisms will out-compete others and as a result, the population in a post-culture environment is radically different than what was on the original sample.

One study analyzed fifteen medicinal cannabis samples using two commercially available culture-based methods. To enumerate and differentiate bacteria and fungi present before and after growth on culture-based media, all samples were further subjected to next-generation sequencing (NGS) and metagenomic analyses (MA). Figure 1 illustrates MA data collected directly from plant material before and after culture on 3M petrifilm and culture-based platforms.

The results demonstrate substantial shifts in bacterial and fungal growth after culturing on the 3M petrifilm and culture-based platforms. Thus, the final composition of microbes after culturing is markedly different from the starting sample. Most concerning is the frequent identification of bacterial species in systems designed for the exclusive quantification of yeast and mold, as quantified by elevated total aerobic count (TAC) Cq values after culture in the total yeast and mold (TYM) medium. The presence of bacterial colonies on TYM growth plates or cartridges may falsely increase the rejection rate of cannabis samples for fungal contamination. These observations call into question the specificity claims of these platforms.

The Live Dead Problem

Figure 2: The enzyme is instantaneously inactivated when lysis buffer is added

One of the common objections to using qPCR for microbial safety testing is the fact that the method does not distinguish between live and dead DNA. PCR primers and probes will amplify any DNA in the sample that matches the target sequence, regardless of viability. Critics claim that this can lead to false positives because DNA from non-viable organisms can inflate results. This is often called the Live-Dead problem. However, scientists have developed multiple solutions to this problem. Most recently, Medicinal Genomics developed the Grim Reefer Free DNA Removal Kit, which eliminates free DNA contained in a sample by simply adding an enzyme and buffer and incubating for 10 minutes. The enzyme is instantaneously inactivated when lysis buffer is added, which prevents the Grim Reefer Enzyme from eliminating DNA when the viable cells are lysed (see Figure 2).

2. Method Must Be Able to Detect Specific Harmful Species 

Toxic Aspergillus spp., which is responsible for at least one confirmed death of a cannabis patient, grows poorly in culture mediums and is severely underreported by current culture-based platforms. And even when Aspergillus does grow in culture, there is a certain non-pathogenic Aspergillus species that look remarkably similar to their pathogenic cousins, making it difficult to speciate using visual identification alone.

Figure 3: The team spiked a known amount of live E. coli into three different environments

Conversely, qPCR assays, such as the PathoSEEK, are designed to target DNA sequences that are unique to pathogenic Aspergillus species, and they can be run using standard qPCR instruments such as the Agilent AriaMx. The primers are so specific that a single DNA base difference in the sequence can determine whether binding occurs. This specificity reduces the frequency of false positives in pathogen detection, a frequent problem with culture-based cannabis testing methods.

Additionally, Medicinal Genomics has developed a multiplex assay that can detect the four pathogenic species of Aspergillus (A. flavus, A. fumigatus, A. niger, and A. terreus) in a single reaction.

3. The Method Must Work on Multiple Matrices 

Figure 4: The team also placed TSB without any E. coli onto a petrifilm to serve as a control.

Marijuana infused products (MIPs) are a very diverse class of matrices that behave very differently than cannabis flowers. Gummy bears, chocolates, oils and tinctures all present different challenges to culture-based techniques as the sugars and carbohydrates can radically alter the carbon sources available for growth. To assess the impact of MIPs on colony-forming units per gram of sample (CFU/g) enumeration, The Medicinal Genomics team spiked a known amount of live E. coli into three different environments: tryptic soy broth (TSB), hemp oil and hard candy. The team then homogenized the samples, pipetted amounts from each onto 3M™ Petrifilm E. coli / Coliform Count (EC) Plates, and incubated for 96 hours. The team also placed TSB without any E. coli onto a petrifilm to serve as a control. Figures 3 and 4 show the results in 24-hour intervals.

Table 1: DNA was spiked into various MIPs

This implies the MIPs are interfering with the reporter assay on the films or that the MIPs are antiseptic in nature.

Many MIPs use citric acid as a flavoring ingredient which may interfere with 3M reporter chemistry. In contrast, the qPCR signal from the Agilent AriaMx was constant, implying there is microbial contamination present on the films, but the colony formation or reporting is inhibited.

Table 3: SenSATIVAx DNA extraction can successfully lyse the cells of the microbes
Table 2: Different numbers of DNA copies spiked into chocolate

This is not an issue with DNA-based methods, so long as the DNA extraction method has been validated on these matrices. For example, the SenSATIVAx DNA extraction method is efficient in different matrices, DNA was spiked into various MIPs as shown in Table 1, and at different numbers of DNA copies into chocolate (Table 2). The SenSATIVAx DNA extraction kit successfully captures the varying levels of DNA, and the PathoSEEK detection assay can successfully detect that range of DNA. Table 3 demonstrates that SenSATIVAx DNA extraction can successfully lyse the cells of the microbes that may be present on cannabis for a variety of organisms spiked onto cannabis flower samples.

Swetha Kaul, PhD

Colorado vs. California: Two Different Approaches to Mold Testing in Cannabis

By Swetha Kaul, PhD
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Swetha Kaul, PhD

Across the country, there is a patchwork of regulatory requirements that vary from state to state. Regulations focus on limiting microbial impurities (such as mold) present in cannabis in order for consumers to receive a safe product. When cultivators in Colorado and Nevada submit their cannabis product to laboratories for testing, they are striving to meet total yeast and mold count (TYMC) requirements.In a nascent industry, it is prudent for state regulators to reference specific testing methodologies so that an industry standard can be established.

TYMC refers to the number of colony forming units present per gram (CFU/g) of cannabis material tested. CFU is a method of quantifying and reporting the amount of live yeast or mold present in the cannabis material being tested. This number is determined by plating the sample, which involves spreading the sample evenly in a container like a petri dish, followed by an incubation period, which provides the ideal conditions for yeast and mold to grow and multiply. If the yeast and mold cells are efficiently distributed on a plate, it is assumed that each live cell will give rise to a single colony. Each colony produces a visible spot on the plate and this represents a single CFU. Counting the numbers of CFU gives an accurate estimate on the number of viable cells in the sample.

The plate count methodology for TYMC is standardized and widely accepted in a variety of industries including the food, cosmetic and pharmaceutical industries. The FDA has published guidelines that specify limits on total yeast and mold counts ranging from 10 to 100,000 CFU/g. In cannabis testing, a TYMC count of 10,000 is commonly used. TYMC is also approved by the AOAC for testing a variety of products, such as food and cosmetics, for yeast and mold. It is a fairly easy technique to perform requiring minimal training, and the overall cost tends to be relatively low. It can be utilized to differentiate between dead and live cells, since only viable living cells produce colonies.

Petri dish containing the fungus Aspergillus flavus
Petri dish containing the fungus Aspergillus flavus.
Photo courtesy of USDA ARS & Peggy Greb.

There is a 24 to 48-hour incubation period associated with TYMC and this impedes speed of testing. Depending on the microbial levels in a sample, additional dilution of a cannabis sample being tested may be required in order to count the cells accurately. TYMC is not species-specific, allowing this method to cover a broad range of yeast and molds, including those that are not considered harmful. Studies conducted on cannabis products have identified several harmful species of yeast and mold, including Cryptococcus, Mucor, Aspergillus, Penicillium and Botrytis Cinerea. Non-pathogenic molds have also been shown to be a source of allergic hypersensitivity reactions. The ability of TYMC to detect only viable living cells from such a broad range of yeast and mold species may be considered an advantage in the newly emerging cannabis industry.

After California voted to legalize recreational marijuana, state regulatory agencies began exploring different cannabis testing methods to implement in order to ensure clean cannabis for the large influx of consumers.

Unlike Colorado, California is considering a different route and the recently released emergency regulations require testing for specific species of Aspergillus mold (A. fumigatus, A. flavus, A. niger and A. terreus). While Aspergillus can also be cultured and plated, it is difficult to differentiate morphological characteristics of each species on a plate and the risk of misidentification is high. Therefore, positive identification would require the use of DNA-based methods such as polymerase chain reaction testing, also known as PCR. PCR is a molecular biology technique that can detect species-specific strains of mold that are considered harmful through the amplification and analysis of DNA sequences present in cannabis. The standard PCR testing method can be divided into four steps:

  1. The double stranded DNA in the cannabis sample is denatured by heat. This refers to splitting the double strand into single strands.
  2. Primers, which are short single-stranded DNA sequences, are added to align with the corresponding section of the DNA. These primers can be directly or indirectly labeled with fluorescence.
  3. DNA polymerase is introduced to extend the sequence, which results in two copies of the original double stranded DNA. DNA polymerases are enzymes that create DNA molecules by assembling nucleotides, the building blocks of DNA.
  4. Once the double stranded DNA is created, the intensity of the resulting fluorescence signal can uncover the presence of specific species of harmful Aspergillus mold, such as fumigatus.

These steps can be repeated several times to amplify a very small amount of DNA in a sample. The primers will only bind to the corresponding sequence of DNA that matches that primer and this allows PCR to be very specific.

PCR testing is used in a wide variety of applications
PCR testing is used in a wide variety of applications
Photo courtesy of USDA ARS & Peggy Greb.

PCR is a very sensitive and selective method with many applications. However, the instrumentation utilized can be very expensive, which would increase the overall cost of a compliance test. The high sensitivity of the method for the target DNA means that there are possibilities for a false positive. This has implications in the cannabis industry where samples that test positive for yeast and mold may need to go through a remediation process to kill the microbial impurities. These remediated samples may still fail a PCR-based microbial test due to the presence of the DNA. Another issue with the high selectivity of this method is that other species of potentially harmful yeast and mold would not even be detected. PCR is a technique that requires skill and training to perform and this, in turn, adds to the high overall cost of the test.

Both TYMC and PCR have associated advantages and disadvantages and it is important to take into account the cost, speed, selectivity, and sensitivity of each method. The differences between the two methodologies would lead to a large disparity in testing standards amongst labs in different states. In a nascent industry, it is prudent for state regulators to reference specific testing methodologies so that an industry standard can be established.

Total Yeast & Mold Count: What Cultivators & Business Owners Need to Know

By Parastoo Yaghmaee, PhD
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Editor’s note: This article should serve as a foundation of knowledge for yeast and mold in cannabis. Beginning in January 2018, we will publish a series of articles focused entirely on yeast and mold, discussing topics such as TYMC testing, preventing yeast and mold in cultivation and treatment methods to reduce yeast and mold.

Cannabis stakeholders, including cultivators, extractors, brokers, distributors and consumers, have been active in the shadows for decades. With the legalization of recreational adult use in several states, and more on the way, safety of the distributed product is one of the main concerns for regulators and the public. Currently, Colorado1, Nevada and Canada2 require total yeast and mold count (TYMC) compliance testing to evaluate whether or not cannabis is safe for human consumption. As the cannabis industry matures, it is likely that TYMC or other stringent testing for yeast and mold will be adopted in the increasingly regulated medical and recreational markets.

The goal of this article is to provide general information on yeast and mold, and to explain why TYMC is an important indicator in determining cannabis safety.

Yeast & Mold

Photo credit: Steep Hill- a petri dish of mold growth from tested cannabis

Yeast and mold are members of the fungi family. Fungus, widespread in nature, can be found in the air, water, soil, vegetation and in decaying matter. The types of fungus found in different geographic regions vary based upon humidity, soil and other environmental conditions. In general, fungi can grow in a wide range of pH environments and temperatures, and can survive in harsh conditions that bacteria cannot. They are not able to produce their own food like plants, and survive by breaking down material from their surroundings into nutrients. Mold cannot thrive in an environment with limited oxygen, while yeast is able to grow with or without oxygen. Most molds, if grown for a long enough period, can be detected visually, while yeast growth is usually detected by off-flavor and fermentation.

Due to their versatility, it is rare to find a place or surface that is naturally free of fungi or their spores. Damp conditions, poor air quality and darker areas are inviting environments for yeast and mold growth.

Cannabis plants are grown in both indoor and outdoor conditions. Plants grown outdoors are exposed to wider ranges and larger populations of fungal species compared to indoor plants. However, factors such as improper watering, the type of soil and fertilizer and poor air circulation can all increase the chance of mold growth in indoor environments. Moreover, secondary contamination is a prevalent risk from human handling during harvest and trimming for both indoor and outdoor-grown cannabis. If humidity and temperature levels of drying and curing rooms are not carefully controlled, the final product could also easily develop fungi or their growth by-product.

 What is TYMC?

TYMC, or total yeast and mold count, is the number of colony forming units present per gram of product (CFU/g). A colony forming unit is the scientific means of counting and reporting the population of live bacteria or yeast and mold in a product. To determine the count, the cannabis sample is plated on a petri dish which is then incubated at a specific temperature for three to five days. During this time, the yeast and mold present will grow and reproduce. Each colony, which represents an individual or a group of yeast and mold, produces one spot on the petri dish. Each spot is considered one colony forming unit.

Why is TYMC Measured?

TYMC is an indicator of the overall cleanliness of the product’s life cycle: growing environment, processing conditions, material handling and storage facilities. Mold by itself is not considered “bad,” but having a high mold count, as measured by TYMC, is alarming and could be detrimental to both consumers and cultivators. 

Aspergillus species niger
Photo: Carlos de Paz, Flickr

The vast majority of mold and yeast present in the environment are indeed harmless, and even useful to humans. Some fungi are used commercially in production of fermented food, industrial alcohol, biodegradation of waste material and the production of antibiotics and enzymes, such as penicillin and proteases. However, certain fungi cause food spoilage and the production of mycotoxin, a fungal growth by-product that is toxic to humans and animals. Humans absorb mycotoxins through inhalation, skin contact and ingestion. Unfortunately, mycotoxins are very stable and withstand both freezing and cooking temperatures. One way to reduce mycotoxin levels in a product is to have a low TYMC.

Aspergillus flavus on culture.
Photo: Iqbal Osman, Flickr

Yeast and mold have been found to be prevalent in cannabis in both current and previous case studies. In a 2017 UC Davis study, 20 marijuana samples obtained from Northern California dispensaries were found to contain several yeast and mold species, including Cryptococcus, Mucor, Aspergillus fumigatus, Aspergillus niger, and Aspergillus flavus.3 The same results were reported in 1983, when marijuana samples collected from 14 cannabis smokers were analyzed. All of the above mold species in the 2017 study were present in 13 out of 14 marijuana samples.4

Aspergillus species niger, flavus, and fumigatus are known for aflatoxin production, a type of dangerous mycotoxin that can be lethal.5 Once a patient smokes and/or ingests cannabis with mold, the toxins and/or spores can thrive inside the lungs and body.6, 7 There are documented fatalities and complications in immunocompromised patients smoking cannabis with mold, including patients with HIV and other autoimmune diseases, as well as the elderly.8, 9, 10, 11

For this reason, regulations exist to limit the allowable TYMC counts for purposes of protecting consumer safety. At the time of writing this article, the acceptable limit for TYMC in cannabis plant material in Colorado, Nevada and Canada is ≤10,000 CFU/g. Washington state requires a mycotoxin test.12 California is looking into testing for specific Aspergillus species as a part of their requirement. As the cannabis industry continues to grow and advance, it is likely that additional states will adopt some form of TYMC testing into their regulatory testing requirements.

References:

  1. https://www.colorado.gov/pacific/sites/default/files/Complete%20Retail%20Marijuana%20Rules%20as%20of%20April%2014%202017.pdf
  2. http://laws-lois.justice.gc.ca/eng/acts/f-27/
  3. https://www.ucdmc.ucdavis.edu/publish/news/newsroom/11791
  4. Kagen SL, Kurup VP, Sohnle PG, Fink JN. 1983. Marijuana smoking and fungal sensitization. Journal of Allergy & Clinical Immunology. 71(4): 389-393.
  5. Centre for Disease control and prevention. 2004 Outbreak of Aflatoxin Poisoning – Eastern and central provinces, Kenya, Jan – July 2004. Morbidity and mortality weekly report.. Sep 3, 2004: 53(34): 790-793
  6. Cescon DW, Page AV, Richardson S, Moore MJ, Boerner S, Gold WL. 2008. Invasive pulmonary Aspergillosis associated with marijuana use in a man with colorectal cancer. Diagnosis in Oncology. 26(13): 2214-2215.
  7. Szyper-Kravits M, Lang R, Manor Y, Lahav M. 2001 Early invasive pulmonary aspergillosis in a leukemia patient linked to aspergillus contaminated marijuana smoking. Leukemia Lymphoma 42(6): 1433 – 1437.
  8. Verweii PE, Kerremans JJ, Voss A, F.G. Meis M. 2000. Fungal contamination of Tobacco and Marijuana. JAMA 2000 284(22): 2875.
  9. Ruchlemer R, Amit-Kohn M, Raveh D, Hanus L. 2015. Inhaled medicinal cannabis and the immunocompromised patient. Support Care Cancer. 23(3):819-822.
  10. McPartland JM, Pruitt PL. 1997. Medical Marijuana and its use by the immunocompromised. Alternative Therapies in Health and Medicine. 3 (3): 39-45.
  11. Hamadeh R, Ardehali A, Locksley RM, York MK. 1983. Fatal aspergillosis associated with smoking contaminated marijuana, in a marrow transplant recipient. Chest. 94(2): 432-433.
  12. http://apps.leg.wa.gov/wac/default.aspx?cite=314-55-102